Pichia pastoris yeast cultures comprising reduced antibody-associated variants

ABSTRACT

Methods for producing heterologous proteins are disclosed. In particular, the present disclosure provides improved methods of producing desired proteins, including multi-subunit proteins such as antibodies, with a higher yield and improved purity. In exemplary embodiments, the transformed cells are a yeast, e.g., methylotrophic yeast such as  Pichia pastoris.

RELATED APPLICATION DISCLOSURE

This application is a divisional of U.S. National Phase Applicationsubmitted under 35 U.S.C. 371 based on International Application No.PCT/US14/30453 filed Mar. 17, 2014 (published as WO 2014-145650 on Sep.18, 2014), now U.S. Pat. No. 10,138,294, which claims the benefit ofU.S. Provisional Application Ser. No. 61/791,471, filed Mar. 15, 2013,and also claims the benefit of U.S. Provisional Application Ser. No.61/790,613, filed Mar. 15, 2013, each of which is hereby incorporated byreference in its entirety.

This application includes as part of its disclosure a biologicalsequence listing contained in the file named “43257o3413.txt” and havinga size of 14,286 bytes, created on Mar. 13, 2014, which is herebyincorporated by reference in its entirety.

FIELD

The present disclosure generally relates to methods for producingdesired proteins in yeast and other cells. Included in the disclosureare methods that can be used to express single- and multi-subunitproteins, including antibodies. In exemplary embodiments, the cells area yeast, such as Pichia pastoris. Embodiments of the subject methods canproduce antibodies or other desired proteins with increased yield ascompared to conventional methods. Additionally, this invention isrelated to the area of fermentation. In particular, it relates tofermentation of recombinant yeast cells.

BACKGROUND

Numerous recombinantly produced proteins have received regulatoryapproval for human therapeutic use. A growing number of thesetherapeutic proteins are produced in microbial expression systems.Indeed, according to a recent review, microbial expression systems areused for production of nearly half of the 151 protein-based recombinantpharmaceuticals licensed up to January 2009 by the U.S. Food and DrugAdministration or European Medicines Agency (Ferrer-Miralles et al.,Microbial Cell Factories 2009, 8:17).

Among these recombinantly produced proteins are antibodies, whichconventionally are tetrameric proteins composed of two identical lightchains and two identical heavy chains. Hundreds of therapeuticmonoclonal antibodies (mAbs) are currently either on the market or underdevelopment. The production of functional antibodies generally involvesthe synthesis of the two polypeptide chains as well as a number ofpost-translational events, including proteolytic processing of theN-terminal secretion signal sequence; proper folding and assembly of thepolypeptides into tetramers; formation of disulfide bonds; and typicallyincludes a specific N-linked glycosylation.

The yeast Pichia pastoris has previously been used as a production hostfor the manufacture of recombinant proteins of therapeutic utility.Examples include the production of Human Serum Albumin and theKallikrein inhibitor, Ecallantide (Reichert, J. mAbs 4:3 1-3, 2012).Pichia pastoris has been used for the production of recombinantmonoclonal antibodies having correctly assembled heavy and light chains(U.S. Pat. No. 7,927,863, which is hereby incorporated by reference inits entirety). The glyceraldehyde-3-phosphate dehydrogenase (GAP)promoter can drive expression of an antibody lacking N-glycosylation inyeast (U.S. Pat. No. 7,927,863).

Recent work by Baumann et al. (BMC Genomics 2011, 12:218), using the GAPsystem to produce recombinant antibody Fab fragment has shown that thissystem, previously thought to be constitutive, exhibits increasedexpression under hypoxic conditions using glucose as the source ofcarbon and energy. Hypoxic conditions are those that allow the dissolvedoxygen level in a fermentation to drop to very low levels while stillsupplying oxygen to the culture through aeration and agitation. Thisresults in mixed aerobic and fermentative metabolism. The use of hypoxicconditions in a fermentor can result in the toxic accumulation ofethanol, and care must be exercised to control the process such thattoxic levels do not accumulate. Baumann accomplished this by measuringthe level of ethanol in the fermentor and adjusting the glucose feedrate to reduce its accumulation. However this method is not veryscalable, as technology for reliably measuring ethanol in large scalefermentors is not widely available.

Fungal hosts such as the methylotrophic yeast Pichia pastoris havedistinct advantages for therapeutic protein expression, including thatthey do not secrete high amounts of endogenous proteins, have stronginducible promoters available for producing heterologous proteins, canbe grown in defined chemical media and without the use of animal sera,and can produce high titers of recombinant proteins (Cregg et al., FEMSMicrobiol. Rev. 24: 45-66 (2000)). Prior work, including work conductedby the present inventors, has helped established P. pastoris as acost-effective platform for producing functional antibodies that arcsuitable for research, diagnostic, and therapeutic use. See co-ownedU.S. Pat. Nos. 7,927,863 and 7,935,340, each of which is incorporated byreference herein in its entirety. Methods are also known in theliterature for design of P. pastoris fermentations for expression ofrecombinant proteins, with optimization having been described withrespect to parameters including cell density, broth volume, pH,substrate feed rate, and the length of each phase of the reaction. SeeZhang et al., “Rational Design and Optimization of Fed-Batch andContinuous Fermentations” in Cregg, J. M., Ed., 2007, Pichia Protocols(2nd edition), Methods in Molecular Biology, vol. 389, Humana Press,Totowa, N.J., pgs. 43-63, which is hereby incorporated by reference inits entirety.

Additionally, prior work by the present applicants and others hasdescribed increasing production of proteins in yeast through methodsincluding addition of a bolus of ethanol to the culture at or near thebeginning of the production phase, and with respect to multi-subunitproteins such as antibodies, varying the number of gene copies and thecopy number ratio between subunit genes. See US20130045888, entitled,Multi-Copy Strategy For High-Titer And High-Purity Production OfMulti-Subunit Proteins Such As Antibodies In Transformed Microbes SuchAs Pichia pastoris; and US20120277408, entitled, High-Purity ProductionOf Multi-Subunit Proteins Such As Antibodies In Transformed MicrobesSuch As Pichia pastoris, each of which is hereby incorporated byreference in its entirety.

Though the aforementioned Zhang et al. article makes some effort todescribe a systematic approach to optimizing the aforementionedparameters and give a theoretical approach to understanding theinterplay between some of these parameters, expression optimizationremains a largely empirical process. Because of this interplay, it isgenerally insufficient to optimize each individual parameter whilekeeping all others constant. For example, optimal media composition mayvary with culture density, strain background, feed rate, agitation,oxygenation, etc. Because of this complex interplay, the number ofcombinations of parameters that can be tested is potentially infinite,and even if an expression system has been extensively optimized therealways remain a large number of untested conditions which could benefityield and/or purity.

SUMMARY

As further described below, Applicants have identified methods ofgreatly increasing the yield of desired proteins produced in eukaryoticcells such as yeast. Even for expression systems which had already beenhighly optimized, the subject method increased the yield of desiredprotein by up to about 30%.

In one aspect, the disclosure provides a method of producing a desiredprotein, comprising: (a) culturing eukaryotic cells comprising one ormore genes that provide for the expression of said desired protein at afirst temperature; and (b) culturing said eukaryotic cells at a secondtemperature and allowing said eukaryotic cells to produce said desiredprotein; wherein said second temperature is different than said firsttemperature. Optionally, said culturing may comprise the steps of: (a)culturing under fed-batch fermentation conditions a population of yeastcells in a culture medium, wherein each yeast cell comprises a DNAsegment encoding a polypeptide, wherein said DNA segment is operablylinked to a glyceraldehyde-3-phosphate (GAP) transcription promoter anda transcription terminator, wherein the protein is notglyceraldehyde-3-phosphate, wherein the fermentation comprises afermentable sugar feed at a first feed rate and wherein the fermentationis agitated at a first oxygen transfer rate; (b) measuring respiratoryquotient (RQ) of the population during the batch fermentation anddetermining if it is within a desired predetermined range, wherein thedesired predetermined range of RQ at about 20-40 hours after initiationof the culturing is between about 1.08 and about 1.35; (c) adjusting oneor both of the fermentable sugar feed rate to a second feed rate or theoxygen transfer rate to a second oxygen transfer rate, when the RQ isoutside of a desired predetermined range; (d) repeating steps (b) and(c) one or more times throughout the step of culturing; (e) harvestingthe yeast cells from the culture medium; and (f) recovering thepolypeptide from the cells and/or the culture medium.

Said first temperature may be between about 20 degrees C. and about 32degrees C.

Said first temperature may be between about 24 degrees C. and about 31.5degrees C.

Said first temperature may be between about 27 degrees C. and about 31degrees C.

Said first temperature may be between about 27.5 degrees C. and about 30degrees C.

Said first temperature may be between about 20 degrees C. and about 29.5degrees C.

Said first temperature may be between about 24 degrees C. and about 29degrees C.

Said first temperature may be between about 27 degrees C. and about 28.5degrees C.

Said first temperature may be between about 27.5 degrees C. and about28.5 degrees C.

Said second temperature may be between about 1 degree C. and about 6degrees C. higher than said first temperature.

Said second temperature may be between about 1 degree C. and about 3degrees C. higher than said first temperature.

Said second temperature may be between about 2 degrees C. and about 4degrees C. higher than said first temperature.

Said second temperature may be between about 2 degrees C. and about 3degrees C. higher than said first temperature.

Said second temperature may be between about 30 degrees C. and about 34degrees C.

Said second temperature may be between about 30 degrees C. and about 32degrees C.

Said second temperature may be between about 30 degrees C. and about31.5 degrees C.

Said second temperature may be about 30 degrees C. or about 31 degreesC.

Said second temperature may be higher than said first temperature.

Said desired protein comprises a multi-subunit complex.

Said multi-subunit complex may comprise an antibody.

Said antibody may be human or humanized.

Said antibody may be specific for IL-6, TNFalpha, CGRP, PCSK9, HGF, orNGF.

Said method may increase the yield of said desired protein.

Said method may decrease the relative abundance of one or moreproduct-associated variants relative to the same method effected withouta difference between said first temperature and said second temperature.

Said method may decrease the relative abundance of product-associatedvariants having a higher or lower apparent molecular weight than saiddesired multi-subunit complex as detected by size exclusionchromatography or gel electrophoresis relative to the same methodeffected without a difference between said first temperature and saidsecond temperature.

Said method may decrease the relative abundance of complexes havingaberrant disulfide bonds relative to the same method effected without adifference between said first temperature and said second temperature.

Said method may decrease the relative abundance of complexes havingreduced cysteines relative to the same method effected without adifference between said first temperature and said second temperature.

Said method may decrease the relative abundance of complexes havingaberrant glycosylation relative to the same method effected without adifference between said first temperature and said second temperature.

Said method may decrease the relative abundance of one or moreproduct-associated variants relative to the same method effected withouta difference between said first temperature and said second temperature.

Said eukaryotic cells may comprise yeast cells.

Said yeast cells may comprise methylotrophic yeast.

Said methylotrophic yeast may be of the genus Pichia.

Said methylotrophic yeast of the genus Pichia may be Pichia pastoris.

Said methylotrophic yeast of the genus Pichia may be selected from thegroup consisting of: Pichia angusta, Pichia guilliermondii, Pichiamethanolica, and Pichia inositovera.

The genes that provide for expression of said desired protein may beintegrated into one or more genomic loci.

At least one of said genomic loci may be selected from the groupconsisting of the pGAP locus, 3′ AOX TT locus; PpURA5; OCH1; AOX1; HIS4;GAP; pGAP; 3′ AOX TT; ARG; and the HIS4 TT locus.

At least one of the genes encoding said subunits of said desired proteinmay be expressed under control of an inducible or constitutive promoter.

Said inducible promoter may be selected from the group consisting of theAOX1, CUP1, tetracycline inducible, thiamine inducible, and FLD1promoters.

Step (a) may comprise culturing said eukaryotic cells in a culturemedium comprising glycerol as a carbon source until said glycerol isexhausted.

Said desired protein may be expressed under control of a promoterselected from the group consisting of: the CUP1, AOX1, ICL1,glyceraldehyde-3-phosphate dehydrogenase (GAP), FLD1, ADH1, alcoholdehydrogenase II, GAL4, PHO3, PHO5, and Pyk promoters, tetracyclineinducible promoters, thiamine inducible promoters, chimeric promotersderived therefrom, yeast promoters, mammalian promoters, insectpromoters, plant promoters, reptile promoters, amphibian promoters,viral promoters, and avian promoters.

Said eukaryotic cell may be a diploid, tetraploid cell, or polyploid.

The method may further comprise purifying said desired protein from saideukaryotic cells or from the culture medium.

Said desired protein may be purified from an intracellular component,cytoplasm, nucleoplasm, or a membrane of said eukaryotic cells.

Said eukaryotic cells may secrete said desired protein into the culturemedium.

Step (a) may comprise a batch phase.

Said batch phase may comprise culturing the eukaryotic cell in a mediumcomprising a carbon source.

The end of said batch phase may be determined by exhaustion of thecarbon source in the culture medium.

Step (b) may comprise a fed batch phase.

The respiratory quotient (RQ) may be maintained at a specified value orin a specified range during step (b).

Said specified RQ value may be about 1.12.

Said specified RQ range may be about 1.0 to 1.24, or about 1.06 to 1.18,or about 1.09 to 1.15.

Said RQ value may or said RQ range may be maintained by modulating oneor more of the feed rate, feed composition, supplied air flow rate,agitation rate, and/or oxygen concentration of supplied air.

The method may comprise a batch phase and a fed batch phase.

The batch phase may comprise culturing the eukaryotic cells with acarbon source until said carbon source is depleted. Said carbon sourcemay comprise one or more of: glycerol, glucose, ethanol, citrate,sorbitol, xylose, trehalose, arabinose, galactose, fructose, melibiose,lactose, maltose, rhamnose, ribose, mannose, mannitol, and raffinose,and preferably comprises glycerol.

The fed batch phase may be initiated after the batch phase.

The temperature shift may be effected at or near the end of the batchphase, at or near the beginning of the fed batch phase, between thebatch phase and the fed batch phase.

For example the temperature shift may be effected after depletion of thecarbon source, or prior to commencing feed addition, or subsequent tocommencing feed addition.

Preferably the temperature shift is effected less than 5 hours aftercommencing feed addition, e.g., less than 4 hours, less than 3 hours,less than 2 hours, less than 1 hour, less than 30 minutes, less than 20minutes, or less than 5 minutes after commencing feed addition. However,the temperature shift may be effected at an earlier or later time.

The method may include the addition of an ethanol bolus to the culture.The ethanol bolus may be added concurrently with the exhaustion of thecarbon source added to the initial culture, which may be detected by arapid increase in the dissolved oxygen concentration (dissolved oxygenspike) or by other means.

The ethanol bolus may result in a concentration of ethanol in theculture of between about 0.01% and about 4%, between about 0.02% andabout 3.75%, between about 0.04% and about 3.5%, between about 0.08% andabout 3.25%, between about 0.1% and about 3%, between about 0.2% andabout 2.75%, between about 0.3% and about 2.5%, between about 0.4% andabout 2.25%, between about 0.5% and about 1.5%, between about 0.5% andabout 2%, between about 0.6% and about 1.75%, between about 0.7% andabout 1.5%, or between about 0.8% and about 1.25%.

The ethanol bolus may result in a concentration of ethanol in theculture of that may be at least about 0.01%, 0.02%, 0.03%, 0.04%, 0.05%,0.06%, 0.07%, 0.08%, 0.09%, 0.10%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%,0.8% or 0.9% (w/v).

The ethanol bolus may result in a concentration of ethanol in theculture of that may be at most about 4%, 3.5%, 3%, 2.5%, 2%, 1.8%, 1.6%,1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1.0%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%,0.35%, 0.3%, 0.25%, 0.2%, or 0.15% (w/v).

The step of adding the ethanol bolus may comprise adding ethanol to saidculture, adding a carrier comprising ethanol to said culture, addingsaid cells to a medium or carrier comprising ethanol, or replacing partof the culture medium.

Said desired protein may contain one or more polypeptides comprising atleast one disulfide bond.

Said desired protein may comprise a multi-subunit complex.

Said multi-subunit complex may comprise an antibody.

The method may decrease the relative abundance of one or moreproduct-associated variants relative to the same method effected in theabsence of the temperature shift.

The method may decrease the relative abundance of product-associatedvariants having a higher or lower apparent molecular weight than saiddesired multi-subunit complex as detected by size exclusionchromatography or gel electrophoresis relative to the same methodeffected in the absence of the temperature shift.

The method may decrease the relative abundance of complexes havingaberrant stoichiometry relative to the same method effected in theabsence of the temperature shift.

The method may decrease the relative abundance of complexes havingaberrant disulfide bonds relative to the same method effected in theabsence of the temperature shift.

The method may comprise adding a feed to the eukaryotic cells.

The respiratory quotient (RQ) value may be maintained at a specifiedvalue or in a specified range. Said specified RQ value may be 1.12. Saidspecified RQ range may be, for example, 1.0 to 1.24, or 1.06 to 1.18, or1.09 to 1.15.

The specified RQ value or range may be maintained by modulating(increasing or decreasing) one or more of the concentration of glucose,availability of oxygen, intensity of agitation, gas pressure, flow rateof supplied air or other gas mixture, viscosity of the culture, culturedensity, concentration of oxygen in the supplied air or other gasmixture, and temperature. For example, the rate of feed addition (feedrate) may be modulated (increased or decreased) in order to control therespiratory quotient (RQ), e.g., to maintain a specified RQ value orrange.

Said feed may comprise at least one fermentable carbon source.

Said carbon source may comprise one or more of: glycerol, glucose,ethanol, citrate, sorbitol, xylose, trehalose, arabinose, galactose,fructose, melibiose, lactose, maltose, rhamnose, ribose, mannose,mannitol, and raffinose.

The genes that provide for expression of said desired protein may beintegrated into one or more genomic loci.

At least one of said genomic loci may be selected from the groupconsisting of the pGAP locus, 3′ AOX TT locus; PpURA5; OCH1; AOX1; HIS4;GAP; pGAP; 3′ AOX TT; ARG; and the HIS4 TT locus.

At least one of the genes encoding said subunits of the multi-subunitcomplex may be expressed under control of an inducible or constitutivepromoter.

Said inducible promoter may be selected from the group consisting of theAOX1, CUP1, tetracycline inducible, thiamine inducible, and FLD1promoters.

At least one of the genes encoding said subunits of the multi-subunitcomplex may be expressed under control of a promoter selected from thegroup consisting of: the CUP1, AOX1, ICL1, glyceraldehyde-3-phosphatedehydrogenase (GAP), FLD1, ADH1, alcohol dehydrogenase II, GAL4, PHO3,PHO5, and Pyk promoters, tetracycline inducible promoters, thiamineinducible promoters, chimeric promoters derived therefrom, yeastpromoters, mammalian promoters, insect promoters, plant promoters,reptile promoters, amphibian promoters, viral promoters, and avianpromoters.

Said eukaryotic cell may be a diploid, tetraploid cell, or polyploid.

The method may further comprise purifying said multi-subunit complexfrom said eukaryotic cells or from the culture medium.

Said multi-subunit complex may be purified from an intracellularcomponent, cytoplasm, nucleoplasm, or a membrane of said eukaryoticcells.

Said eukaryotic cells secrete said desired protein into the culturemedium.

Said desired protein may be purified from said culture medium.

Said desired protein may comprise a monospecific or bispecific antibody.

Said desired protein may comprise a human antibody or a humanizedantibody or fragment thereof.

Said humanized antibody may be of mouse, rat, rabbit, goat, sheep, orcow origin.

Said humanized antibody may be of rabbit origin.

Said desired protein may comprise a monovalent, bivalent, or multivalentantibody.

Said antibody may be purified from said culture by protein A and/orprotein G affinity.

At least one of the genes that provide for expression of said desiredprotein may be optimized for expression in said eukaryotic cell.

Said desired protein may comprise an antibody and the purity of saidantibody may be assessed by measuring the fraction of the antibodyproduced by said eukaryotic cell that may be contained in antibodycomplexes having the expected apparent hydrodynamic radius, may becontained in antibody complexes having the expected molecular weight,and/or specifically binds a target of said antibody.

Said desired protein may comprise an antibody and the yield of saidantibody may be assessed by determining the amount of antibody producedby said eukaryotic cell discounting any product-associated variants thatmay be abnormally glycosylated, contained in antibody complexes otherthan complexes having the expected apparent hydrodynamic radius,contained in antibody complexes having the expected molecular weight,and/or that fail to specifically bind to the target of said antibody.

The molecular weight of said antibody complexes may be determined bynon-reducing SDS-PAGE.

Said desired protein may comprise an antibody, said method may furthercomprise purifying said antibody.

Said culture cell may produce a supernatant antibody titer of at least100 mg/L, at least 150 mg/L, at least 200 mg/L, at least 250 mg/L, atleast 300 mg/L, between 100 and 300 mg/L, between 100 and 500 mg/L,between 100 and 1000 mg/L, at least 1000 mg/L, at least 1250 mg/liter,at least 1500 mg/liter, at least about 1750 mg/liter, at least about2000 mg/liter, at least about 10000 mg/liter, or more.

Said desired protein may comprise a multi-subunit complex and one ormore subunits of said multi-subunit complex may be expressed from morethan one gene copy.

Said desired protein may comprise an antibody which may be expressedfrom between 1-10 copies of a gene encoding the light chain of saidantibody and from 1-10 copies of a gene encoding the heavy chain of saidantibody.

The gene(s) that provide for expression of said desired protein may beintegrated into genome of said cells.

The gene(s) that provide for expression of said desired protein may becontained on an extrachromosomal element, plasmid, or artificialchromosome.

Said cells may comprise more copies of the gene that provide for theexpression of the light chain of said antibody than copies of the genethat provide for expression of the heavy chain of said antibody.

The respective number of copies of the gene encoding the heavy chain ofsaid antibody and the number of copies of the gene encoding the lightchain of said antibody in said cells may be: 2 and 2, 2 and 3, 3 and 3,3 and 4, 3 and 5, 4 and 3, 4 and 4, 4 and 5, 4 and 6, 5 and 4, 5 and 5,5 and 6, or 5 and 7.

The respective number of copies of the gene encoding the heavy chain ofsaid antibody and the number of copies of the gene encoding the lightchain of said antibody in said cells may be: 2 and 1, 3 and 1, 4 and 1,5 and 1, 6 and 1, 7 and 1, 8 and 1, 9 and 1, 10 and 1, 1 and 2, 2 and 2,3 and 2, 4 and 2, 5 and 2, 6 and 2, 7 and 2, 8 and 2, 9 and 2, 10 and 2,1 and 3, 2 and 3, 3 and 3, 4 and 3, 5 and 3, 6 and 3, 7 and 3, 8 and 3,9 and 3, 10 and 3, 1 and 4, 2 and 4, 3 and 4, 4 and 4, 5 and 4, 6 and 4,7 and 4, 8 and 4, 9 and 4, 10 and 4, 1 and 5, 2 and 5, 3 and 5, 4 and 5,5 and 5, 6 and 5, 7 and 5, 8 and 5, 9 and 5, 10 and 5, 1 and 6, 2 and 6,3 and 6, 4 and 6, 5 and 6, 6 and 6, 7 and 6, 8 and 6, 9 and 6, 10 and 6,1 and 7, 2 and 7, 3 and 7, 4 and 7, 5 and 7, 6 and 7, 7 and 7, 8 and 7,9 and 7, 10 and 7, 1 and 8, 2 and 8, 3 and 8, 4 and 8, 5 and 8, 6 and 8,7 and 8, 8 and 8, 9 and 8, 10 and 8, 1 and 9, 2 and 9, 3 and 9, 4 and 9,5 and 9, 6 and 9, 7 and 9, 8 and 9, 9 and 9, 10 and 9, 1 and 10, 2 and10, 3 and 10, 4 and 10, 5 and 10, 6 and 10, 7 and 10, 8 and 10, 9 and10, 10 and 10.

Using the methods of the present disclosure, the relative abundance ofundesired side-product(s) may be decreased by at least 10%, at least20%, at least 30%, at least 40%, at least 50%, at least 60%, at least70%, at least 80%, at least 90%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, or down to undetectable levels comparedto initial abundance levels, relative to conventional methods. Exemplaryundesired side-products whose relative abundance may be so decreased mayinclude one or more species having a different apparent molecular weightthan the desired multi-subunit complex. For example, apparent molecularweight may be affected by differences in stoichiometry, folding, complexassembly, and/or glycosylation. For example, such undesired sideproducts may be detected using size exclusion chromatography and/or gelelectrophoresis, and may have a higher or lower apparent molecularweight than the desired multi-subunit complex. In exemplary embodiments,the undesired side-products may be detected under reducing conditions.In other exemplary embodiments, the undesired side-products may bedetected under non-reducing conditions.

In exemplary embodiments, the present disclosure also provides improvedmethods and compositions of matter that provide for the recombinantproduction of antibodies and other multi-subunit complexes, with ahigher yield. In exemplary embodiments, the yield may be increased by atleast 10%, at least 20%, at least 30%, at least 40%, at least 50%, atleast 100%, or more (relative to conventional methods) using the methodsdisclosed herein.

In exemplary embodiments, the eukaryotic cell in which the desiredprotein may be produced may be a yeast, for example in a Pichia speciessuch as P. pastoris or another methylotrophic yeast, or in aSaccharomyces species such as S. cerevisiae, or another yeast such as aSchizosaccharomyces (e.g., S. pombe). Other examples of methylotrophicyeast which may be utilized in the present invention include Pichiaangusta (also known in the art as Hansenula polymorpha), Pichiaguilliermondii, Pichia methanolica, Pichia inositovera, Ogataeanitratoaversa, and Candida boidnii.

The eukaryotic cell may be a yeast cell, such as a methylotrophic yeast,such as a yeast of the genus Pichia. Exemplary methylotrophic yeasts ofthe genus Pichia include Pichia pastoris, Pichia angusta, Pichiaguilliermondii, Pichia methanolica, and Pichia inositovera. The hostcell may be produced by mating, e.g., by mating two haploid yeast cellsthat each contain one or more copies of at least one gene encoding asubunit of the multi-subunit complex.

In a preferred embodiment, the methylotrophic yeasts of the genus Pichiais Pichia pastoris. The eukaryotic cell may be a haploid, diploid ortetraploid cell.

At least one of the genes encoding said desired protein may be expressedunder control of an inducible or constitutive promoter, such as CUP1(induced by the level of copper in the medium; see Koller et al., Yeast2000; 16: 651-656), tetracycline inducible promoters (see, e.g., Staibet al., Antimicrobial Agents And Chemotherapy, January 2008, p.146-156), thiamine inducible promoters, AOX1, ICL1,glyceraldehyde-3-phosphate dehydrogenase (GAP), FLD1, ADH1, alcoholdehydrogenase II, GAL4, PHO3, PHO5, and Pyk promoters, chimericpromoters derived therefrom, yeast promoters, mammalian promoters,insect promoters, plant promoters, reptile promoters, amphibianpromoters, viral promoters, and avian promoters.

The eukaryotic cell may secrete said desired protein into the culturemedium. For example, said desired protein may comprise a secretionsignal peptide. Alternatively or in addition, said desired multi-subunitcomplex may be retained in said host cell and may be isolated therefrom.

The desired protein may comprise an antibody, such as a monospecific orbispecific antibody. The antibody may be an antibody that specificallybinds any antigen.

The desired protein may comprise an antibody of any type. Exemplaryantibody types include antibodies of any mammalian species, e.g., human,mouse, rat, rabbit, goat, sheep, cow, etc. Preferably, the antibody is ahuman antibody or a humanized antibody that may be of rabbit origin. Theantibody may be a monovalent, bivalent, or multivalent antibody.

At least one of said genes that provide for expression of the desiredprotein, such as the light chain and/or heavy chain of a desiredantibody, in said eukaryotic cell may be optimized for expression insaid host cell (e.g., by selecting preferred codons and/or altering thepercentage AT through codon selection).

The purity of said desired protein, such as a desired antibody, may beassessed by measuring the fraction of the desired protein produced bysaid host cell that is non-glycosylated, is contained in complexeshaving the expected apparent hydrodynamic radius and/or apparentmolecular weight (e.g., measured by size exclusion chromatography), hasthe expected electrophoretic mobility (e.g., detected by gelelectrophoresis, such as SDS-PAGE, and optionally Western blotting),and/or by measuring the specific activity of the multi-subunit complex(e.g., specific binding a target of a desired antibody).

The desired protein may be an antibody, and yield of said antibody maybe assessed by determining the amount of desired antibody produced bysaid host cell discounting any product-associated variants that areglycosylated, contained in antibody complexes other than complexeshaving the expected apparent molecular weight or hydrodynamic radius,and/or that fail to specifically bind to the target of said desiredantibody.

According to another embodiment of the invention a method is providedthat produces a desired protein such as an antibody or antigen-bindingfragment of an antibody in yeast cells. A population of yeast cells iscultured under fed-batch fermentation conditions in a culture medium.Each yeast cell comprises a DNA segment encoding a desired protein, suchas a heavy chain polypeptide, and optionally a DNA segment encoding asecond desired protein, such as a light chain polypeptide of anantibody. The one or more DNA segments are operably linked to aglyceraldehyde-3-phosphate (GAP) transcription promoter and atranscription terminator. The fermentation comprises a fermentable sugarfeed at a first feed rate, and the fermentation is oxygenated at a firstoxygen transfer rate. The respiratory quotient (RQ) of the population ismeasured during the feeding phase of the fed-batch fermentation and itis compared to a desired predetermined range. One or both of thefermentable sugar feed rate and the oxygen transfer rate are adjusted toa second rate when the RQ is outside of a desired predetermined range.The measuring and adjusting are performed throughout all or part of theculturing. The yeast cells are harvested from the culture medium.Desired proteins, such as heavy chain and light chain polypeptidesproduced by the yeast cells, are recovered from yeast cell-depletedculture medium or from the yeast cells.

According to another embodiment of the invention a method is providedfor producing an antibody comprising two heavy chains and two lightchains in Pichia yeast cells. A population of Pichia yeast cells iscultured under hypoxic, fed-batch fermentation conditions in a culturemedium. Each yeast cell comprises a DNA segment encoding a heavy chainpolypeptide and a DNA segment encoding a light chain polypeptide of anantibody. DNA segments are operably linked to aglyceraldehyde-3-phosphate (GAP) transcription promoter and atranscription terminator. The yeast cells are harvested from the culturemedium. The antibody produced by the yeast cells is recovered from theyeast cells or from the yeast cell-depleted culture medium.

The present disclosure also provides a feedback control mechanism for afermentation of yeast cells to make desired proteins that uses arespiratory quotient measurement which adjusts the levels of oxygenationand/or fermentable sugar feed. The feedback control mechanism permitswell controlled cultures that produce good amounts of product whileavoiding toxic accumulation of ethanol. Additionally, desired proteinsso produced have excellent qualitative properties, such as excellenthomogeneity and proper inter-subunit assembly. For example, thedisclosure provides method for producing a desired protein, in yeastcells, comprising the steps of: (a) culturing under fed-batchfermentation conditions a population of yeast cells in a culture medium,wherein each yeast cell comprises a DNA segment encoding a polypeptide,wherein said DNA segment is operably linked to aglyceraldehyde-3-phosphate (GAP) transcription promoter and atranscription terminator, wherein the protein is notglyceraldehyde-3-phosphate, wherein the fermentation comprises afermentable sugar feed at a first feed rate and wherein the fermentationis agitated at a first oxygen transfer rate; (b) measuring respiratoryquotient (RQ) of the population during the batch fermentation anddetermining if it is within a desired predetermined range, wherein thedesired predetermined range of RQ at about 20-40 hours after initiationof the culturing is between about 1.08 and about 1.35; (c) adjusting oneor both of the fermentable sugar feed rate to a second feed rate or theoxygen transfer rate to a second oxygen transfer rate, when the RQ isoutside of a desired predetermined range; (d) repeating steps (b) and(c) one or more times throughout the step of culturing; (e) harvestingthe yeast cells from the culture medium; and (f) recovering thepolypeptide from the cells and/or the culture medium. Optionally, saidmethod may include (a) culturing eukaryotic cells comprising one or moregenes that provide for the expression of said desired protein at a firsttemperature; and (b) culturing said eukaryotic cells at a secondtemperature and allowing said eukaryotic cells to produce said desiredprotein; wherein said second temperature may be different than saidfirst temperature; for example, said first temperature may be betweenabout 20 degrees C. and about 32 degrees C., between about 24 degrees C.and about 31.5 degrees C., between about 27 degrees C. and about 31degrees C., between about 27.5 degrees C. and about 30 degrees C.,between about 20 degrees C. and about 29.5 degrees C., between about 24degrees C. and about 29 degrees C., between about 27 degrees C. andabout 28.5 degrees C., or between about 27.5 degrees C. and about 28.5degrees C.; and/or optionally said second temperature may be betweenabout 1 degree C. and about 6 degrees C. higher than said firsttemperature, between about 1 degree C. and about 3 degrees C. higherthan said first temperature, between about 2 degrees C. and about 4degrees C. higher than said first temperature, or between about 2degrees C. and about 3 degrees C. higher than said first temperature;and/or optionally said second temperature may be between about 30degrees C. and about 34 degrees C., between about 30 degrees C. andabout 32 degrees C., or between about 30 degrees C. and about 31.5degrees C.; such as said first temperature may be about 28 degrees C.and said second temperature may be about 30 degrees C. or about 31degrees C.; or said first temperature may be between about 27.5 degreesC. and about 28.5 degrees C. and said second temperature may be betweenabout 30 degrees C. and about 31 degrees C.; wherein optionally saidfirst temperature is higher than said second temperature.

Said step (d) may be performed at intervals of between about 1 and 5minutes, such as at intervals of about 3 minutes.

Said step (d) may be performed continuously.

At least one adjustment to the feed rate may be made in step (c).

Step (c) may be performed automatically using a feedback controlmechanism linked to a device which measures the RQ.

The yeast cells may be from a species selected from the group consistingof Pichia pastoris, Pichia methanolica, Pichia angusta, Pichiathermomethanolica, and Saccharomyces cerevisiae.

The method may further comprise delivering ethanol to the yeast cells atabout 10 to 14 hours of the culturing to achieve a level in thefermentation of about 8.0 to about 12.0 g/l ethanol.

The desired predetermined range of RQ at about 20-40 hours afterinitiation of the culturing may be between about 1.09 and about 1.25.

The step of harvesting may be performed at about 80-110 hours after theinitiation of the culturing.

The ethanol concentration may be measured during the step of culturing,and adjustments may be made to stabilize the ethanol concentration aboveabout 5 g/l and below about 25 g/l, wherein said adjustments may be madeby adjusting one or both of the fermentable sugar feed rate to a thirdfeed rate or the oxygen transfer rate to a third oxygen transfer rate.For example, said adjustments may be made to stabilize the ethanolconcentration above about 5 g/l and below about 17 g/l.

The desired predetermined range of RQ at 20-110 hours of thefermentation may be selected from the group consisting of: about1.08-1.1; about 1.08-1.15; about 1.08-1.2; about 1.08-1.25; about1.08-1.3; and about 1.08-1.35.

Step (b) of measuring may be performed by sampling the exhaust gas ofthe fermentation.

Step (b) of measuring may be performed using a mass spectrometer,infrared analyzer, or paramagnetic analyzer.

In step (c) the oxygen transfer rate may be adjusted, either byincreasing the oxygen transfer rate when the RQ may be too high ordecreasing the oxygen transfer rate when the RQ may be too low.

In step (c) the fermentable sugar feed rate may be adjusted, either byincreasing the fermentable sugar feed rate when the RQ may be too low ordecreasing the fermentable sugar feed rate when the RQ may be too high.

In step (c) the fermentable sugar feed rate may be also adjusted, eitherby increasing the fermentable sugar feed rate when the RQ may be too lowand decreasing the fermentable sugar feed rate when the RQ may be toohigh.

Step (c) may be performed by modulating agitation rate of the culture.

The DNA segment may encode an antibody heavy chain or an antibody lightchain, or a fragment of an antibody.

The polypeptide may be harvested from the culture medium.

Each yeast cell may comprise a DNA segment encoding a heavy chainpolypeptide and a DNA segment encoding a light chain polypeptide of anantibody.

In another aspect, the disclosure provides a method for producing anantibody comprising two heavy chains and two light chains or an antibodyfragment in Pichia yeast cells, comprising the steps of: (a) culturingunder hypoxic, fed-batch fermentation conditions a population of Pichiayeast cells in a culture medium, wherein each yeast cell comprises a DNAsegment encoding a heavy chain polypeptide and DNA segment encoding alight chain polypeptide of an antibody, wherein said DNA segments may beoperably linked to a glyceraldehyde-3-phosphate (GAP) transcriptionpromoter and a transcription terminator; (b) harvesting the yeast cellsfrom the culture medium; and (c) recovering the antibody produced by theyeast cells from the yeast cell-depleted culture medium. Optionally,said method may include (a) culturing eukaryotic cells comprising one ormore genes that provide for the expression of said desired protein at afirst temperature; and (b) culturing said eukaryotic cells at a secondtemperature and allowing said eukaryotic cells to produce said desiredprotein; wherein said second temperature may be different than saidfirst temperature; for example, said first temperature may be betweenabout 20 degrees C. and about 32 degrees C., between about 24 degrees C.and about 31.5 degrees C., between about 27 degrees C. and about 31degrees C., between about 27.5 degrees C. and about 30 degrees C.,between about 20 degrees C. and about 29.5 degrees C., between about 24degrees C. and about 29 degrees C., between about 27 degrees C. andabout 28.5 degrees C., or between about 27.5 degrees C. and about 28.5degrees C.; and/or optionally said second temperature may be betweenabout 1 degree C. and about 6 degrees C. higher than said firsttemperature, between about 1 degree C. and about 3 degrees C. higherthan said first temperature, between about 2 degrees C. and about 4degrees C. higher than said first temperature, or between about 2degrees C. and about 3 degrees C. higher than said first temperature;and/or optionally said second temperature may be between about 30degrees C. and about 34 degrees C., between about 30 degrees C. andabout 32 degrees C., or between about 30 degrees C. and about 31.5degrees C.; such as said first temperature may be about 28 degrees C.and said second temperature may be about 30 degrees C. or about 31degrees C.; or said first temperature may be between about 27.5 degreesC. and about 28.5 degrees C. and said second temperature may be betweenabout 30 degrees C. and about 31 degrees C.; wherein optionally saidfirst temperature is higher than said second temperature.

The Pichia yeast cells may be selected from the group consisting ofPichia pastoris, Pichia methanolica, Pichia angusta, and Pichiathermomethanolica. For example, the Pichia yeast cells may be Pichiapastoris.

In another aspect, the disclosure provides a large scale fermentationprocess comprising the steps of: (i) culturing yeast cells underlarge-scale, fed-batch fermentation conditions, wherein said culturedyeast cells may be engineered to express a desired protein; (ii)periodically or continuously monitoring the RQ values during thefed-batch fermentation; and determining whether the RQ value fallswithin a specified range; (iii) adjusting at least one culture parameterat least once during the fed-batch fermentation so as to adjust ormaintain the RQ value of the fed-batch yeast culture whereby it may bewithin the specified range; and (iv) harvesting the yeast cells or theculture medium and recovering the desired protein from the harvestedcells or culture medium of step (iii). Optionally, said method mayinclude (a) culturing eukaryotic cells comprising one or more genes thatprovide for the expression of said desired protein at a firsttemperature; and (b) culturing said eukaryotic cells at a secondtemperature and allowing said eukaryotic cells to produce said desiredprotein; wherein said second temperature may be different than saidfirst temperature; for example, said first temperature may be betweenabout 20 degrees C. and about 32 degrees C., between about 24 degrees C.and about 31.5 degrees C., between about 27 degrees C. and about 31degrees C., between about 27.5 degrees C. and about 30 degrees C.,between about 20 degrees C. and about 29.5 degrees C., between about 24degrees C. and about 29 degrees C., between about 27 degrees C. andabout 28.5 degrees C., or between about 27.5 degrees C. and about 28.5degrees C.; and/or optionally said second temperature may be betweenabout 1 degree C. and about 6 degrees C. higher than said firsttemperature, between about 1 degree C. and about 3 degrees C. higherthan said first temperature, between about 2 degrees C. and about 4degrees C. higher than said first temperature, or between about 2degrees C. and about 3 degrees C. higher than said first temperature;and/or optionally said second temperature may be between about 30degrees C. and about 34 degrees C., between about 30 degrees C. andabout 32 degrees C., or between about 30 degrees C. and about 31.5degrees C.; such as said first temperature may be about 28 degrees C.and said second temperature may be about 30 degrees C. or about 31degrees C.; or said first temperature may be between about 27.5 degreesC. and about 28.5 degrees C. and said second temperature may be betweenabout 30 degrees C. and about 31 degrees C.; wherein optionally saidfirst temperature is higher than said second temperature.

The protein may be an antibody protein or antibody fragment.

The protein (e.g., antibody protein or antibody fragment) may besecreted by the yeast.

The adjusted culture parameters may include one or more of (a) air flowrate, b) oxygen concentration, (c) feed composition, (d) feed rate, (e)cell density, and (f) agitation.

The adjusted culture parameter may comprise one or more of (a) the feedcomposition, (b) the feed rate and (c) the oxygen transfer rate; andwherein the culture parameter may be adjusted at least once during theprocess so as to adjust or maintain the RQ value of the fed-batch yeastculture such that it may be within the specified range.

The method may further include a batch fermentation preceding thefed-batch fermentation culture.

The fed-batch fermentation may be conducted for at least 50 hours, orfor at least 70 hours.

The yeast cells may be Pichia pastoris, such as polyploid, haploid, ordiploid Pichia pastoris.

The cell density of the fed-batch culture may comprise from 1 to 700 g/lwet cell weight.

In step (iii) the feed rate may be increased or decreased so as toadjust the RQ value to fall within the specified range.

In step (iii) the feed composition may be altered by altering the amountof at least one fermentable sugar or other hydrocarbon so as to adjustthe RQ value to fall within the specified range.

In step (iii) the amount of oxygen in the fed-batch fermentation may beincreased or decreased during the fed-batch fermentation so as to adjustthe RQ value to fall within the specified range.

In step (iii) wherein the amount of at least one fermentable sugar orother fermentable hydrocarbon may be increased or decreased so as toadjust the RQ value to fall within the specified range.

In step (iii) the feed rate may be increased or decreased during thefed-batch fermentation so as to adjust the RQ value to fall within thespecified range.

The process may result in an improvement in a property selected fromantibody purity and antibody production relative to a fed-batch processwhich does not include step (iii).

The specified range for the RQ value in step (iii) may be between about1.08-1.35, or between about 1.08-1.2.

These and other embodiments which will be apparent to those of skill inthe art upon reading the specification, provide the art with methods andproducts with improved reliability, predictability, efficiency, andquality.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: shows the RQ profiles of 3 different Mab1 Strains A, B, and C.The vertical line indicates the time at which RQ control was initiated.

FIG. 2: shows the Agitation profiles of 3 different Mab1 Strains A, B,and C. The vertical line indicates the time at which RQ control wasinitiated.

FIG. 3: shows the Ethanol profiles of 3 different Mab1 Strains A, B, andC.

FIG. 4: shows the Growth profiles of 3 different Mab1 Strains A, B, andC.

FIG. 5: shows the Whole Broth Titer profiles of 3 different Mab1 StrainsA, B, and C.

FIG. 6: shows the RQ profiles of Mab1 Strain A at 3 different RQ Setpoints RQ 1.09-1.15, 1.19-1.25, 1.29-1.35. The vertical line indicatesthe time at which RQ control was initiated.

FIG. 7: shows the Agitation profiles of Mab1 Strain A at 3 different RQSet points RQ 1.09-1.15, 1.19-1.25, 1.29-1.35. The vertical lineindicates the time at which RQ control was initiated.

FIG. 8: shows the Ethanol profiles of Mab1 Strain A at 3 different RQSet points RQ 1.09-1.15, 1.19-1.25, 1.29-1.35.

FIG. 9: shows the Growth profiles of Mab1 Strain A at 3 different RQ Setpoints RQ 1.09-1.15, 1.19-1.25, 1.29-1.35.

FIG. 10: shows the Whole Broth Titer profiles of Mab1 Strain A at 3different RQ Set points RQ 1.09-1.15, 1.19-1.25, 1.29-1.35.

FIG. 11: shows the RQ profiles of Mab1 Strain A grown under aerobic andhypoxic conditions. The vertical line indicates the time at which RQcontrol was initiated.

FIG. 12: shows the Agitation profiles of Mab1 Strain A grown underaerobic and hypoxic conditions. The vertical line indicates the time atwhich RQ control was initiated.

FIG. 13: shows the Ethanol profiles of Mab1 Strain A grown under aerobicand hypoxic conditions.

FIG. 14: shows the Growth profiles of Mab1 Strain A grown under aerobicand hypoxic conditions.

FIG. 15: shows the % DO profiles of Mab1 Strain A grown under aerobicand hypoxic conditions.

FIG. 16: shows the Whole Broth Titer profiles of Mab1 Strain A grownunder aerobic and hypoxic conditions.

FIG. 17: shows the RQ profiles of 4 different Mab2 Strains A, B, C andD. The vertical line indicates the time at which RQ control wasinitiated.

FIG. 18: shows the Agitation profiles of 4 different Mab2 Strains A, B,C and D strains. The vertical line indicates the time at which RQcontrol was initiated.

FIG. 19: shows the Ethanol profiles of 4 different Mab2 Strains A, B, Cand D strains.

FIG. 20: shows the Growth profiles of 4 different Mab2 Strains A, B, Cand D strains.

FIG. 21: shows the Whole Broth Titer profiles of 4 different Strains A,B, C and D strains.

FIG. 22: shows the RQ profiles of 3 different Mab3 Strains A, B, and C.The vertical line indicates the time at which RQ control was initiated.

FIG. 23: shows the Agitation profiles of 3 different Mab3 Strains A, B,and C. The vertical line indicates the time at which RQ control wasinitiated.

FIG. 24: shows the Ethanol profiles of 3 different Mab3 Strains A, B,and C.

FIG. 25: shows the Growth profiles of 3 different Mab3 Strains A, B, andC.

FIG. 26: shows the Whole Broth Titer profiles of 3 different Mab3Strains A, B, and C.

FIG. 27: shows the RQ profiles of Mab3 Strain B at different feed rates.The vertical line indicates the time at which RQ control was initiated.

FIG. 28: shows the Agitation profiles of Mab3 Strain B at different feedrates. The vertical line indicates the time at which RQ control wasinitiated.

FIG. 29: shows the Ethanol profiles of Mab3 Strain B at different feedrates.

FIG. 30: shows the Growth profiles of Mab3 Strain B at different feedrates.

FIG. 31: shows the Whole Broth Titer profiles of Mab3 Strain B atdifferent feed rates.

FIG. 32: shows the RQ profiles of Mab4 Strain A at different bed rates.The vertical line indicates the time at which RQ control was initiated.

FIG. 33: shows the Agitation profiles of Mab4 Strain A at different feedrates. The vertical line indicates the time at which RQ control wasinitiated.

FIG. 34: shows the Ethanol profiles of Mab4 Strain A strain at differentfeed rates.

FIG. 35: shows the Growth profiles of Mab4 Strain A strain at differentfeed rates.

FIG. 36: shows the Whole Broth profiles of Mab4 Strain A strain atdifferent feed rates.

FIG. 37: shows the Whole Broth profiles of Mab2 Strain A for the aerobic(B1) and hypoxic (B2) process in large scale fermentation.

FIG. 38: shows the SDS-PAGE Non-Reduced and Reduced gels of Mab1 StrainA at fermentation times 62, 70, and 86 hours for aerobic and hypoxicprocess conditions.

FIG. 39: shows the SDS-PAGE Non-Reduced and Reduced gels of Mab1 StrainA at fermentation times 62, 69, and 85 hours.

FIG. 40 illustrates the beneficial effect of a culture temperature shifton recombinant antibody production. Whole broth (“WB”) antibody titer(arbitrary units) is plotted against time points throughout fermentationfor five different temperature shifts and an unshifted control(maintained at 28° C.). Pre-shift temperature was 28° C. and post-shifttemperature was 25° C., 29.5° C., 31° C., 32.5° C., or 34° C. asindicated. Upward temperature shifts of between 1.5° C. and 3° C. (finaltemperature, 29.5° C. and 31° C., respectively) resulted in increasedfinal titers. The antibody produced in these experiment was Ab-A. Forthe culture shifted to 31° C., the final titer was increased by about30% relative to the unshifted control culture (maintained at 28° C.).

FIG. 41A-B summarizes purity of the recombinant antibodies produced witha culture temperature shift in FIG. 40. Purity was assessed by sizeexclusion chromatography of protein A-purified antibody harvested at theend of antibody production. Panel A shows the purity assessed undernon-reducing conditions. Peaks were detected corresponding to the fullantibody (“Main peak IgG”) and two aberrant antibody complexes (“PrepeakHHL” and “75 kD HL”). Numbers shown are percentage of the total detectedprotein contained in each peak. A similar proportion of the totalprotein was contained in the main antibody peak for unshifted cultures(i.e., maintained at 28° C.) and cultures shifted to 29.5° C. and 31° C.Panel B shows purity detected under reduced conditions. For eachtemperature condition the percentage of total detected protein containedin the heavy chain (“HC”), light chain (“LC”) and the total heavy andlight chain protein (“Total HC+LC”) is shown, along with the percentagecontained in three other peaks (“RT 9.80,” “RT 10.16,” and “RT 10.80”).As compared to the unshifted culture maintained at 28° C., thepercentage of protein contained in the heavy and light chain peaksremained similar for the culture shifted to 29.5° C. and was increasedby about 4% for higher temperature shifts.

FIG. 42A-C details purity of the recombinant antibodies produced asshown in FIG. 40 for the culture shifted downward to 25° C. Panel A,size exclusion chromatography traces and tabulated results fornon-reduced samples. Panel B, Coomassie stained gel electrophoresisresults for non-reduced (lane 1) and reduced (lane 3) samples. Lanes 2and 4 show a size marker. Panel 3, size exclusion chromatography tracesand tabulated results for reduced samples. Chromatography results aretabulated and summarized in FIG. 41.

FIG. 43A-C details purity of the recombinant antibody as shown in FIG.40 for the control culture produced without a temperature shift andmaintained at 28° C. Panel A, size exclusion chromatography traces andtabulated results for non-reduced samples. Panel B, Coomassie stainedgel electrophoresis results for non-reduced (lane 1) and reduced (lane3) samples. Lanes 2 and 4 show a size marker. Panel 3, size exclusionchromatography traces and tabulated results for reduced samples.Chromatography results are tabulated and summarized in FIG. 41.

FIG. 44A-C details purity of the recombinant antibodies produced asshown in FIG. 40 for the culture shifted upward by 1.5° C. to 29.5° C.Panel A, size exclusion chromatography traces and tabulated results fornon-reduced samples. Panel B, Coomassie stained gel electrophoresisresults for non-reduced (lane 1) and reduced (lane 3) samples. Lanes 2and 4 show a size marker. Panel 3, size exclusion chromatography tracesand tabulated results for reduced samples. Chromatography results aretabulated and summarized in FIG. 41.

FIG. 45A-C details purity of the recombinant antibodies produced asshown in FIG. 40 for the culture shifted upward by 3° C. to 31° C. PanelA, size exclusion chromatography traces and tabulated results fornon-reduced samples. Panel B, Coomassie stained gel electrophoresisresults for non-reduced (lane 1) and reduced (lane 3) samples. Lanes 2and 4 show a size marker. Panel 3, size exclusion chromatography tracesand tabulated results for reduced samples. Chromatography results aretabulated and summarized in FIG. 41.

FIG. 46A-C details purity of the recombinant antibodies produced asshown in FIG. 40 for the culture shifted upward by 4.5° C. to 32.5° C.Panel A, size exclusion chromatography traces and tabulated results fornon-reduced samples. Panel B, Coomassie stained gel electrophoresisresults for non-reduced (lane 1) and reduced (lane 3) samples. Lanes 2and 4 show a size marker. Panel 3, size exclusion chromatography tracesand tabulated results for reduced samples. Chromatography results aretabulated and summarized in FIG. 41.

FIG. 47A-C details purity of the recombinant antibodies produced asshown in FIG. 40 for the culture shifted upward by 6° C. to 34° C. PanelA, size exclusion chromatography traces and tabulated results fornon-reduced samples. Panel B, Coomassie stained gel electrophoresisresults for non-reduced (lane 1) and reduced (lane 3) samples. Lanes 2and 4 show a size marker. Panel 3, size exclusion chromatography tracesand tabulated results for reduced samples. Chromatography results aretabulated and summarized in FIG. 41.

FIG. 48A-B shows improvement in titer of Ab-B resulting from atemperature shift during culture. Panel A: whole broth antibody titer(arbitrary units) is shown graphically versus time in culture of ahigh-expressing strain comprising 4 copies of the Ab-B heavy chain geneand 3 copies of the Ab-B light chain gene (“H4/L3”). Thetemperature-shifted culture (“H4/L3 30C”) exhibited a greater titer atall time-points than the two non-shifted cultures (“H4/L3 28C”). Theaverage final antibody titer was increased by about 28%. Panel B: wholebroth antibody titer (arbitrary units) is shown graphically versus timein a culture of a lower-expressing strain comprising 3 copies of theAb-B heavy chain gene and 3 copies of the Ab-B light chain gene(“H3/L3”). The temperature-shifted culture (“H3/L3 30C”) exhibited agreater titer than the average of the two non-shifted cultures (“H3/L328C”).

FIG. 49 summarizes the purity of the recombinant antibodies producedwith a culture temperature shift as shown in FIG. 48. Purity wasassessed by size exclusion chromatography of protein A-purified antibodyharvested at the end of antibody production. Labels are as described inFIG. 41.

FIG. 50 shows improvement in titer of Ab-A resulting from a temperatureshift during culture. Whole broth antibody titer (arbitrary units) isplotted versus time for cultures which were subjected to a temperatureshift during culture or control cultures for which a temperature shiftwas not performed. Four cultures were shifted from 28° C. to 30° C.after initiating feed addition (label “30C”) and five control cultureswere unshifted and maintained at 28° C. throughout culturing. Each ofthe shifted cultures exhibited higher titers than the non-shiftedcultures. The average increase in titer resulting from the temperatureshift was about 47%.

FIG. 51 summarizes the purity of the recombinant antibodies producedwith a culture temperature shift as shown in FIG. 50. Purity wasassessed by size exclusion chromatography of protein A-purified antibodyharvested at the end of antibody production. Labels are as described inFIG. 41.

FIG. 52 shows the shows the temperature of each culture in Example 13plotted versus time in culture.

DETAILED DESCRIPTION

This disclosure describes methods of improving the yield and/or purityof recombinantly expressed proteins, including antibodies and othermulti-subunit proteins. Methods are provided wherein a temperature shiftis effected during cell culture. The inclusion of a temperature shift isdemonstrated below to improve the yield and purity of recombinantlyexpressed antibodies, compared to expression in the absence of theshift.

Though not intending to be limited by theory, it is hypothesized that atemperature shift can cause sustained changes in gene expression whichconfer a lasting improvement in recombinant protein production. Saidimprovement may be mediated by improvements in protein expression,stability, folding, post-translational processing, and (in the case ofantibodies and other multi-subunit complexes) proper subunit assembly,e.g., due to a sustained increase in expression of heat shock proteins.

Preferred host cells include yeasts, and particularly preferred yeastsinclude methylotrophic yeast strains, e.g., Pichia pastoris, Hansenulapolymorpha (Pichia angusta), Pichia guilliermondii, Pichia methanolica,Pichia inositovera, and others (see, e.g., U.S. Pat. Nos. 4,812,405,4,818,700, 4,929,555, 5,736,383, 5,955,349, 5,888,768, and 6,258,559each of which is incorporated by reference in its entirety). The hostcell may be produced by methods known in the art, such astransformation, mating, sporulation, etc.

In exemplary embodiments, the disclosure provides methods which decreasethe production of one or more undesired side products. Relative to thedesired protein, the undesired side product(s) may exhibit one or moreof: altered stoichiometry (in the case of a multi-subunit complex),aberrant glycosylation, differences in apparent molecular weight,differences in disulfide bonds, differences in hydrodynamic radius,fragments and/or truncations. Undesired side-products may exhibit one ormore additional differences as well. Undesired side-products may also bedetected by their effects on a preparation, e.g., alteration in thelevel of specific activity, immunogenicity, or other effects on physicalconstitution and/or function of the desired multi-subunit complex.

For example, when the desired protein is an antibody, the undesired sideproducts may include an H1L1 or “half antibody” species (i.e.,containing a heavy chain and a light chain, wherein the heavy chain isnot linked by a disulfide bond to another heavy chain), and/or a H2L1species (i.e., containing two heavy chains and one light chain, butlacking a second light chain).

In a preferred embodiment, the host cell may comprise more than one copyof one or more of the gene encoding the desired protein or the genesencoding the subunits thereof. For example, multiple copies of a subunitgene may be integrated in tandem into one or more chromosomal loci.Tandemly integrated gene copies are preferably retained in a stablenumber of copies during culture for the production of the multi-subunitcomplex. For example, a co-owned application published as US2013/0045888 describes experiments wherein gene copy numbers weregenerally stable for P. pastoris strains containing three to fourtandemly integrated copies of light and heavy chain antibody genes.

One or more of the genes encoding the desired protein may be integratedinto one or multiple chromosomal loci of a host cell. Any suitablechromosomal locus may be utilized for integration, including intergenicsequences, promoters sequences, coding sequences, termination sequences,regulatory sequences, etc. Exemplary chromosomal loci that may be usedin P. pastoris include PpURA5; OCH1; AOX1; HIS4; and GAP. The encodinggenes may also be integrated into one or more random chromosomal locirather than being targeted. In exemplary embodiments, the chromosomalloci are selected from the group consisting of the pGAP locus, 3′ AOXTT, and the HIS4 TT locus. In additional exemplary embodiments, thegenes encoding the heterologous protein subunits may be contained in oneor more extrachromosomal elements, for example one or more plasmids orartificial chromosomes.

In exemplary embodiments, the desired protein may be a multi-subunitcomplex comprising two, three, four, five, six, or more identical ornon-identical subunits. Additionally, each subunit may be present one ormore times in each multi-subunit protein. For example, the desiredprotein may comprise a multi-specific antibody such as a bi-specificantibody comprising two non-identical light chains and two non-identicalheavy chains. As a further example, the desired protein may comprise anantibody comprising two identical light chains and two identical heavychains.

The subunits may be expressed from monocistronic genes, polycistronicgenes, or any combination thereof. Each polycistronic gene may comprisemultiple copies of the same subunit, or may comprise one or more copiesof each different subunit.

Exemplary methods that may be used for manipulation of Pichia pastoris(including methods of culturing, transforming, and mating) are disclosedin Published Applications including U.S. 20080003643, U.S. 20070298500,and U.S. 20060270045, and in Higgins, D. R., and Cregg, J. M., Eds.1998. Pichia Protocols. Methods in Molecular Biology. Humana Press,Totowa, N.J., and Cregg, J. M., Ed., 2007, Pichia Protocols (2ndedition), Methods in Molecular Biology. Humana Press, Totowa, N.J., eachof which is incorporated by reference in its entirety.

An exemplary expression cassette that may be utilized is composed of theglyceraldehyde dehydrogenase gene (GAP gene) promoter, fused tosequences encoding a secretion signal, followed by the sequence of thegene to be expressed, followed by sequences encoding a P. pastoristranscriptional termination signal from the P. pastoris alcohol oxidaseI gene (AOX1). The Zeocin resistance marker gene may provide a means ofenrichment for strains that contain multiple integrated copies of anexpression vector in a strain by selecting for transformants that areresistant to higher levels of Zeocin. Similarly, G418 or Kanamycinresistance marker genes may be used to provide a means of enrichment forstrains that contain multiple integrated copies of an expression vectorin a strain by selecting for transformants that are resistant to higherlevels of Geneticin or Kanamycin.

Host strains that may be utilized include auxotrophic P. pastoris orother Pichia strains, for example, strains having mutations in met1,lys3, ura3 and ade1 or other auxotrophy-associated genes. Preferredmutations are incapable of giving rise to revertants at any appreciablefrequency and are preferably partial or even more preferably fulldeletion mutants. For example, prototrophic diploid or tetraploidstrains are produced by mating a complementing sets of auxotrophicstrains. Said strains have the advantage of being able to be grown onminimal media, and additionally said media tend to select against growthof haploid cells that may arise through sporulation.

Transformation of haploid and diploid P. pastoris strains and geneticmanipulation of the P. pastoris sexual cycle may be performed asdescribed in Pichia Protocols (1998, 2007), supra.

Prior to transformation, each expression vector may be linearized byrestriction enzyme cleavage within a region homologous to the targetgenomic locus (e.g., the GAP promoter sequence) to direct theintegration of the vectors into the target locus in the host cell.Samples of each vector may then be individually transformed intocultures of the desired strains by electroporation or other methods, andsuccessful transformants may be selected by means of a selectablemarker, e.g., antibiotic resistance or complementation of an auxotrophy.Isolates may be picked, streaked for single colonies under selectiveconditions and then examined to confirm the number of copies of the geneencoding the desired protein or subunit of the multi-subunit complex(e.g., a desired antibody) by Southern Blot or PCR assay on genomic DNAextracted from each strain. Optionally, expression of the expectedsubunit gene product may be confirmed, e.g., by FACS, Western Blot,colony lift and immunoblot, and other means known in the art.Optionally, isolates are transformed additional times to introduceadditional heterologous genes, e.g., additional copies of the geneencoding a desired protein, or in the case of a multi-subunit protein,genes encoding the same subunit may be integrated at a different locus,and/or copies of a different subunit may be integrated. Strains may beproduced as haploids, diploids, or other ploidies (including tetraploidand higher ploidies). Haploid strains may be produced and mated in orderto rapidly test different combinations of gene copy numbers, e.g.,numbers of copies of a single gene encoding a desired protein, ornumbers of copies of the differing genes encoding the subunits of amulti-subunit protein. Presence of the desired protein gene or eachexpected subunit gene may be confirmed by Southern blotting, PCR, andother detection means known in the art. Additionally, expression of anantibody or other desired protein may also be confirmed by a colonylift/immunoblot method (Wung et al. Biotechniques 21 808-812 (1996))and/or by FACS.

Transformation is optionally repeated to target a heterologous gene intoa second locus, which may be the same gene or a different gene than wastargeted into the first locus. When the construct to be integrated intothe second locus encodes a protein that is the same as or highly similarto the sequence encoded by the first locus, its sequence may be variedto decrease the likelihood of undesired integration into the firstlocus. Such sequence differences may also promote genetic stability bydecreasing the likelihood of subsequent recombination events. Forexample, the sequence to be integrated into the second locus may havedifferences in the promoter sequence, termination sequence, codon usage,and/or other tolerable sequence differences relative to the sequenceintegrated into the first locus.

To mate P. pastoris haploid strains, each strain to be crossed can bepatched together onto mating plates. For example, multiple matings canbe conveniently performed at the same time by streaking each strain tobe mated across a plate suitable for its growth, and the mating partnersmay be streaked across a second plate (preferably the plates are richmedia such as YPD). Typically, after one or two days incubation at 30°C., cells from the two plates can be replica plated in a crisscrossfashion onto a mating plate, resulting in a cross-hatched pattern witheach pair of strains being co-plated and having the opportunity to mateat the intersection of a pair of the original streak lines. The matingplate can then be incubated (e.g., at 30° C.) to stimulate theinitiation of mating between strains. After about two days, the cells onthe mating plates can be streaked, patched, or replica plated onto mediaselective for the desired diploid strains (e.g., where the mated strainshave complementary autotrophies, drop-out or minimal medium plates maybe used). These plates can be incubated (e.g., at 30° C.) for a suitableduration (e.g., about three days) to allow for the selective growth ofthe desired diploid strains. Colonies that arise can be picked andstreaked for single colonies to isolate and purify each diploid strain.

Expression vectors for use in the methods of the invention may furtherinclude yeast specific sequences, including a selectable auxotrophic ordrug marker for identifying transformed yeast strains. A drug marker mayfurther be used to amplify copy number of the vector in a yeast hostcell, e.g., by culturing a population of cells in an elevatedconcentration of the drug, thereby selecting transformants that expresselevated levels of the resistance gene.

In an exemplary embodiment, one or more of the genes encoding theheterologous protein subunits are coupled to an inducible promoter.Suitable exemplary promoters include the alcohol oxidase 1 genepromoter, formaldehyde dehydrogenase genes (FLD; see U.S. Pub. No.2007/0298500), and other inducible promoters known in the art. Thealcohol oxidase 1 gene promoter, is tightly repressed during growth ofthe yeast on most common carbon sources, such as glucose, glycerol, orethanol, but is highly induced during growth on methanol (Tschopp etal., 1987; U.S. Pat. No. 4,855,231 to Stroman, D. W., et al). Forproduction of foreign proteins, strains may be initially grown on arepressing carbon source to generate biomass and then shifted tomethanol as the sole (or main) carbon and energy source to induceexpression of the foreign gene. One advantage of this regulatory systemis that P. pastoris strains transformed with foreign genes whoseexpression products are toxic to the cells can be maintained by growingunder repressing conditions.

In another exemplary embodiment, one or more of the heterologous genesmay be coupled to a regulated promoter, whose expression level can beupregulated under appropriate conditions. Exemplary regulated promotersinclude the CUP1 promoter (induced by the level of copper in themedium), tetracycline inducible promoters, thiamine inducible promoters,the AOX1 promoter, and the FLD1 promoter.

In another aspect, the disclosure provides a process for making desiredproteins in yeast which may employ a particular type of feedback controlmechanism to increase the productivity of fermentations. That feedbackcontrol mechanism allows the robust and precise control of mixed aerobicand fermentative metabolism that stimulates optimal production of thedesired product. This can be used to good effect to produce recombinantproteins such as monoclonal antibodies in yeast, particularly in Pichiapastoris, and more particularly using the glyceraldehyde-3-phosphate(GAP) promoter.

The process using the feedback control mechanism is applicable to theproduction of full-length, correctly assembled recombinant monoclonalantibodies, as well as to antibody fragments and other recombinantproteins, i.e., not glyceraldehyde-3-phosphate. The control mechanismthat we employ is easy to mechanize and render automatic, thuseliminating much labor in monitoring and adjusting fermentationconditions. The process is applicable to production of a variety ofantibodies and other desired proteins and is readily scalable toaccommodate commercial, e.g., large scale, production needs.

The production process may use Respiratory Quotient (RQ) as a feedbackcontrol variable. RQ can be used to balance mass transfer parametersand/or fermentable sugar feed rate in order to maintain a hypoxic statein the culture while preventing the toxic accumulation of ethanol, aby-product of fermentative metabolism. RQ is defined as the molar rateof carbon dioxide produced divided by the molar rate of oxygen consumedin the culture. It can be measured by analyzing the exhaust gas comingfrom the fermentor for content of carbon dioxide and oxygen. Thismetabolic parameter can be measured continuously or intermittentlythroughout the desired growth phase using readily available means.Examples of appropriate intervals for measurements are hourly,half-hourly, quarter-hourly, ten minutes, five minutes, four minutes,three minutes, two minutes, one minute. Time periods during measurementsmay vary with growth conditions, from initiating the culture throughharvest. Exemplary periods for measurement and control are between 20and 40 hours, between 10 and 60 hours, between 5 and 70 hours, andbetween 20 and 110 hours after initiating of the culturing in thefermentor.

When yeast cells are grown in a completely anaerobic state, without thepresence of oxygen, they are said to be using fermentative metabolism toproduce the energy they need to grow. In this case the followingstoichiometric equation for the conversion of glucose to ethanolapplies:C₆H₁₂O₆⇒2C₂H₅OH+2CO₂+H₂O+Energy

When yeast cells obtain their energy solely from aerobic metabolism ofglucose, then oxygen is consumed, and only carbon dioxide and water areproduced. In the presence of oxygen, yeast cells use aerobic metabolism,which is more efficient, i.e., more energy is obtained from a mole ofglucose under aerobic metabolism than under fermentative metabolism.

The RQ of a culture producing only ethanol from glucose approachesinfinity (since no oxygen is consumed, the denominator of RQ is zero),whereas for purely aerobic metabolism of glucose the RQ approaches thevalue of 1.0 (three moles of oxygen are consumed to produce 3 moles ofcarbon dioxide). Thus values higher than 1 indicate a mixed metaboliccondition where both aerobic and fermentative metabolism are takingplace simultaneously. Typically oxygen transfer rate and/or fermentablesugar feed rate can be adjusted using RQ as a feedback control variableto accomplish this mixed metabolism. Using such a mixed metabolism,hypoxic conditions can be maintained. A hypoxic state exists when thereis a low level of fermentative metabolism controlled by the equilibriumof oxygen transfer rate and fermentable sugar feed rate. Hypoxicconditions may be defined by an RQ above 1.0 with dissolved oxygen belowabout 5%.

RQ can be measured in the exhaust gas stream from a fermentor. Any knownand suitable method for ascertaining the molar concentration of oxygenconsumed and carbon dioxide generated can be used. Exemplary techniqueswhich may be used are mass spectrometry, infrared spectroscopy, andparamagnetic analysis.

Hypoxic growth has a beneficial effect on the production of full length,properly assembled proteins, such as antibodies, in Pichia. We tried toreduce the dissolved oxygen concentration simply by reduction of theagitator speed during fermentation. However, it was not possible toobtain reliable control in this manner, because small differences inagitation rate or fermentable sugar feed rate would quickly result inthe accumulation of toxic levels of ethanol.

A feedback control mechanism can also be used to measure and controlethanol levels through modulation of either fermentable sugar feed rateand/or oxygen transfer rate, e.g., by agitator speed. Controllingaccumulation of ethanol should permit a more stable process. In order tomonitor ethanol levels one can use a probe inserted into the fermentor.The probe can monitor ethanol levels in the fermentation brothcontinuously. However, it is not feasible to use such a probe incommercial manufacturing of molecules under Good ManufacturingProcesses, because it does not have an output that can be sufficientlycalibrated.

When RQ is maintained in a narrow range from approximately 1.1 toapproximately 2, ethanol accumulation stabilizes at levels that are nottoxic. Preferably the concentration of ethanol is maintained betweenabout 5 g/I and 17 g/I. Moreover, these same conditions stimulate theGAP promoter, leading to significantly increased desired protein, e.g.,antibody production over aerobic fermentation conditions. RQ ranges thatmay be desirable include about 1.08-2.0; about 1.08-1.85; about1.08-1.65; about 1.08-1.45; about 1.08-1.35; about 1.08-1.25; about1.08-1.2; and about 1.08-1.15. Alternative carbon sources other thanglucose can achieve an RQ less than 1. Such carbon sources includeacetate and glycerol. Other suitable RQ ranges include 1.08 to 1.35, and1.15 to 1.25. RQ can be monitored and controlled during any desiredportion of the fermentation, for example from 0 to 110 hours, from 20-40hours, from 20-70 hours, from 20-90 hours, from 20-110 hours, or anyother desired time period.

Thus RQ can be manipulated and changed over time by addition of variouscarbon sources, by addition of various amounts of a carbon source, andby manipulation of the oxygen levels. In one embodiment, oxygen levelsare manipulated by increasing or decreasing agitation. In anotherembodiment, the ratio of oxygen to nitrogen gas in the gas feed iscontrolled. Ways that the oxygen transfer rate can be adjusted includethe changing the air flow rate, the oxygen concentration, the celldensity, the temperature, and agitation. In another embodiment glucoseor other fermentable sugar feed is modulated to affect the RQ. Otherfermentable sugars which can be used in the feed include withoutlimitation fructose, sucrose, maltose, and maltotriose. Feed rate orcomposition can be modulated to affect the RQ. The control of RQ may bemanual or automatic.

Protein-encoding nucleic acids, e.g., encoding antibodies, may be on asingle or multiple continuous or discontinuous segments of a recombinantconstruct. Antibodies may be any type of fragment or construct or fulllength. These may be, for example, Fab, F(ab′)₂, Fe, and ScFv. In someembodiments, the chains and or chain fragments will assemble properly invivo. If assembly is not proper, in vitro assembly may be necessary.Other proteins which may be desirably made are those having one or moresubunits, whether heteromeric or homomeric. Typically the protein willbe useful for diagnostic or therapeutic purposes. The protein may be agrowth factor, a cytokine, a blood coagulation factor, a therapeutictoxin, a structural protein useful for reconstruction, an enzyme, etc.

Proteins such as antibodies may be recovered from the cell-depletedculture medium or from the cells by any technique known in the art.Typically a binding step will be used to reduce the volume of thepreparation. Binding can be done on filters or columns or other solidsupports, as is convenient. In some embodiments, protein A may be usedas an antibody capturing agent. The protein A may be bound to apolymeric matrix.

Any type of yeast cells can be used, including Saccharomyces, Hansenula,and Pichia species. Exemplary but not limiting species which may be usedare P. pastoris, P. methanolica, P. angusta, P. thermomethanolica,Hansenula polymorpha, and S. cerevisiae. The yeast may be haploid ordiploid.

Other promoters like GAP may be used similarly. These are typicallypromoters that are for genes that are up-regulated in hypoxic,glucose-limiting growth in yeast cells, such as Pichia. Such promoterswhich may be used include, without limitation, promoters for genesYHR140W, YNL040W, NTA1, SGT1, URK1, PGI1, YHR112C, CPS1, PET18, TPA1,PFK1, SCS7, YIL166C, PFK2, HSP12, ERO1, ERG11, ENO1, SSP120, BNA1, DUG3,CYS4, YEL047C, CDC19, BNA2, TDH3, ERG28, TSA1, LCB5, PLB3, MUP3, ERV14,PDX3, NCP1, TPO4, CUS1, COX15, YBR096W, DOG1, YDL124W, YMR244W, YNL134C,YEL023C, PIC2, GLK1, ALD5, YPR098C, ERG1, HEM13, YNL200C, DBP3, HAC1,UGA2, PGK1, YBR056W, GEF1, MTD1, PDR16, HXT6, AQR1, YPL225W, CYS3, GPM1,THI11, UBA4, EXG1, DGK1, HEM14, SCO1, MAK3, ZRT1, YPL260W, RSB1, AIM19,YET3, YCR061W, EHT1, BAT1, YLR126C, MAE1, PGC1, YHL008C, NCE103, MIH1,ROD1, FBA1, SSA4, PIL1, PDC1-3, THI3, SAM2, EFT2, and INO1.

Large scale fermentation processes are those typically used incommercial processes to produce a useful product. Typically these aregreater than 100 liters in volume. Fed-batch fermentation is a processby which nutrients are added during the fermentation to affect celldensity and product accumulation.

Disclosures of prior published patent applications and patents, U.S.Pat. Nos. 7,927,863, 8,268,582, U.S. App 2012/0277408 are expresslyincorporated herein.

Though much of the present disclosure describes production ofantibodies, the methods described herein are readily adapted to otherdesired proteins including single subunit and multi-subunit proteins.Additionally, the present methods are not limited to production ofmulti-protein complexes but may also be readily adapted for use withribonucleoprotein (RNP) complexes including telomerase, hnRNPs,Ribosomes, snRNPs, signal recognition particles, prokaryotic andeukaryotic RNase P complexes, and any other complexes that containmultiple distinct protein and/or RNA subunits. The host cell thatexpresses a multi-subunit complex may be produced by methods known inthe art. For example, a panel of diploid or tetraploid yeast cellscontaining differing combinations of gene copy numbers may be generatedby mating cells containing varying numbers of copies of the individualsubunit genes (which numbers of copies preferably are known in advanceof mating).

Definitions

It is to be understood that this invention is not limited to theparticular methodology, protocols, cell lines, animal species or genera,and reagents described, as such may vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to limit the scope ofthe present invention which will be limited only by the appended claims.

As used herein the singular forms “a”, “and”, and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “a cell” includes a plurality of such cells andreference to “the protein” includes reference to one or more proteinsand equivalents thereof known to those skilled in the art, and so forth.All technical and scientific terms used herein have the same meaning ascommonly understood to one of ordinary skill in the art to which thisinvention belongs unless clearly indicated otherwise.

Bolus addition: In the present disclosure, “bolus addition” generallyrefers to rapid change in concentration of a substance (such as ethanol)in contact with cultured cells (for example, in a culture medium). Forexample, the substance may be added to the cultured cells in a singleaddition, a succession of more than one addition, and/or infused over aperiod of time (e.g., over about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20,25, 30, 40, 50, 60, 90, or 120 minutes). The substance may also be addedby replacing the culture medium in part or in full, for example byconcentrating the cells (using centrifugation, filtration, settling, orother methods), removing part or all of the medium, and adding thesubstance, or by adding the cells to a medium containing the substance.The substance may be admixed with a carrier (e.g., culture media, water,saline, etc.). For example, a bolus addition of ethanol may comprise theaddition of pure or concentrated ethanol (e.g., 100%, 95%, 70%, 50%,60%, 40%, 30%, 20%, etc.) to the culture medium in an amount sufficientto produce the desired concentration. As another example, the cells maybe added to a medium containing ethanol, e.g., by adding an inoculumcontaining the cells to a medium containing ethanol.

Bolus concentration: In the present disclosure, “bolus concentration”generally refers to the concentration that results from a bolus additionof a substance (e.g., ethanol).

Mating competent yeast species: In the present invention this isintended to broadly encompass any diploid or tetraploid yeast which canbe grown in culture. Such species of yeast may exist in a haploid,diploid, or other polyploid form. The cells of a given ploidy may, underappropriate conditions, proliferate for an indefinite number ofgenerations in that form. Diploid cells can also sporulate to formhaploid cells. Sequential mating can result in tetraploid strainsthrough further mating or fusion of diploid strains. The presentinvention contemplates the use of haploid yeast, as well as diploid orother polyploid yeast cells produced, for example, by mating or fusion(e.g., spheroplast fusion).

In one embodiment of the invention, the mating competent yeast is amember of the Saccharomycetaceae family, which includes the generaArxiozyma; Ascobotryozyma; Citeromyces; Debaryomyces; Dekkera;Eremothecium; Issatchenkia; Kazachstania; Kluyveromyces; Kodamaea;Lodderomyces; Pachysolen; Pichia; Saccharomyces; Saturnispora;Tetrapisispora; Torulaspora; Williopsis; and Zygosaccharomyces. Othertypes of yeast potentially useful in the invention include Yarrowia;Rhodosporidium; Candida; Hansenula; Filobasium; Sporidiobolus; Bullera;Leucosporidium and Filobasidella.

In a preferred embodiment of the invention, the mating competent yeastis a member of the genus Pichia or is another methylotroph. In a furtherpreferred embodiment of the invention, the mating competent yeast of thegenus Pichia is one of the following species: Pichia pastoris, Pichiamethanolica, and Hansenula polymorpha (Pichia angusta). In aparticularly preferred embodiment of the invention, the mating competentyeast of the genus Pichia is the species Pichia pastoris.

Haploid Yeast Cell: A cell having a single copy of each gene of itsnormal genomic (chromosomal) complement.

Polyploid Yeast Cell: A cell having more than one copy of its normalgenomic (chromosomal) complement.

Diploid Yeast Cell: A cell having two copies (alleles) of essentiallyevery gene of its normal genomic complement, typically formed by theprocess of fusion (mating) of two haploid cells.

Tetraploid Yeast Cell: A cell having four copies (alleles) ofessentially every gene of its normal genomic complement, typicallyformed by the process of fusion (mating) of two diploid cells.Tetraploids may carry two, three, four, or more different expressioncassettes. Such tetraploids might be obtained in S. cerevisiae byselective mating homozygotic heterothallic a/a and alpha/alpha diploidsand in Pichia by sequential mating of haploids to obtain auxotrophicdiploids. For example, a [met his] haploid can be mated with [ade his]haploid to obtain diploid [his]; and a [met arg] haploid can be matedwith [ade arg] haploid to obtain diploid [arg]; then the diploid [his]can be mated with the diploid [arg] to obtain a tetraploid prototroph.It will be understood by those of skill in the art that reference to thebenefits and uses of diploid cells may also apply to tetraploid cells.

Yeast Mating: The process by which two yeast cells fuse to form a singleyeast cell. The fused cells may be haploid cells or cells of higherploidy (e.g., mating two diploid cells to produce a tetraploid cell).

Meiosis: The process by which a diploid yeast cell undergoes reductivedivision to form four haploid spore products. Each spore may thengerminate and form a haploid vegetatively growing cell line.

Selectable Marker: A selectable marker is a gene or gene fragment thatconfers a growth phenotype (physical growth characteristic) on a cellreceiving that gene as, for example through a transformation event. Theselectable marker allows that cell to survive and grow in a selectivegrowth medium under conditions in which cells that do not receive thatselectable marker gene cannot grow. Selectable marker genes generallyfall into several types, including positive selectable marker genes suchas a gene that confers on a cell resistance to an antibiotic or otherdrug, temperature when two temperature sensitive (“ts”) mutants arecrossed or a is mutant is transformed; negative selectable marker genessuch as a biosynthetic gene that confers on a cell the ability to growin a medium without a specific nutrient needed by all cells that do nothave that biosynthetic gene, or a mutagenized biosynthetic gene thatconfers on a cell inability to grow by cells that do not have the wildtype gene; and the like. Suitable markers include but are not limitedto: ZEO; NEO (G418); LYS3; MET1; MET3a; ADE1; ADE3; URA3; and the like.

Integrated: A genetic element (typically a heterologous genetic element)that are covalently joined into a chromosome of an organism.

Tandemly integrated: Two or more copies of a genetic element that areintegrated in adjacent locations in a chromosome. The two or more copiesdo not necessarily have the orientation; e.g., for transcribed genes,some copies may be transcribed from the Watson strand and others fromthe Crick strand.

Host cell: In the context of the present disclosure, the term host cellrefers to a cell (e.g., a eukaryotic cell, such as a Pichia cell) whichcontains a heterologous gene. For example, the heterologous gene mayprovide for the expression of a subunit of a desired multi-subunitcomplex, a gene involved in protein folding (e.g., a chaperone),expression, or secretion, and/or another desired gene. The heterologousgene may be integrated into the genome of the eukaryotic cell orcontained in extrachromosomal element such as a plasmid or artificialchromosome.

The respiratory quotient (RQ), is defined as the number of moles of CO₂produced divided by the number of moles of O₂ consumed. For the completeoxidation of carbohydrates, the RQ value is 1.0. Fermentation isindicated by RQ values greater than one, and with no or low O₂utilization (e.g., for metabolism in the absence of molecular oxygen) RQmay reach very large or theoretically infinite values.

Expression Vector: These DNA vectors contain elements that facilitatemanipulation for the expression of a foreign protein within the targethost cell. Conveniently, manipulation of sequences and production of DNAfor transformation is first performed in a bacterial host, e.g. E. coliand usually vectors will include sequences to facilitate suchmanipulations, including a bacterial origin of replication andappropriate bacterial selection marker. Selection markers encodeproteins necessary for the survival or growth of transformed host cellsgrown in a selective culture medium. Host cells not transformed with thevector containing the selection gene will not survive in the culturemedium. Typical selection genes encode proteins that (a) conferresistance to antibiotics or other toxins, (b) complement auxotrophicdeficiencies, or (c) supply critical nutrients not available fromcomplex media. Exemplary vectors and methods for transformation of yeastare described, for example, in Burke, D., Dawson, D., & Stearns, T.(2000). Methods in yeast genetics: a Cold Spring Harbor Laboratorycourse manual. Plainview, N.Y.: Cold Spring Harbor Laboratory Press,which is incorporated by reference herein in its entirety.

Expression vectors for use in the methods of the invention may furtherinclude yeast specific sequences, including a selectable auxotrophic ordrug marker for identifying transformed yeast strains. A drug marker mayfurther be used to select for amplification of copy number of the vectorin a yeast host cell.

The polypeptide coding sequence of interest is typically operably linkedto transcriptional and translational regulatory sequences that providefor expression of the polypeptide in yeast cells. These vectorcomponents may include, but are not limited to, one or more of thefollowing: an enhancer element, a promoter, and a transcriptiontermination sequence. Sequences for the secretion of the polypeptide mayalso be included, e.g. a signal sequence, and the like. A yeast originof replication is optional, as expression vectors are often integratedinto the yeast genome.

Though optional, in one embodiment of the invention, the desired proteinor a subunit of the desired multi-subunit complex is operably linked, orfused, to a secretion sequence that provides for secretion of theexpressed polypeptide into the culture media, which can facilitateharvesting and purification of the desired protein or complex. Even morepreferably, the secretion sequences provide for optimized secretion ofthe polypeptide from the host cells (e.g., yeast diploid cells), such asthrough selecting preferred codons and/or altering the percentage ATthrough codon selection. It is known in the art that secretionefficiency and/or stability can be affected by the choice of secretionsequence and the optimal secretion sequence can vary between differentproteins (see, e.g., Koganesawa et al., Protein Eng. 2001 September;14(9):705-10, which is incorporated by reference herein in itsentirety). Many potentially suitable secretion signals are known in theart and can readily be tested for their effect upon yield and/or purityof a particular desired protein or complex. Any secretion sequences maypotentially be used, including those present in secreted proteins ofyeasts and other species, as well as engineered secretion sequences.Exemplary secretion signal sequences that may be utilized include:chicken lysozyme (CLY) signal peptide (MRSLLILVLCFLPLAALG (SEQ IDNO:5)), CLY-L8 (MRLLLLLLLLPLAALG (SEQ ID NO:6)), S. cerevisiae invertase(SUC2) signal peptide (MLLQAFLFLLAGFAAKISA (SEQ ID NO:7)), MF-alpha(Prepro) (MRFPSIFTAVLFAASSALA-APVNTTE-EGVSLEKR (SEQ ID NO:8)), MF-alpha(Pre)-apv (MRFPSIFTAVLFAASSALA-APV (SEQ ID NO:9)), MF-alpha(Pre)-apv-SLEKR (MRFPSIFTAVLFAASSALA-APVSLEKR (SEQ ID NO:10)), MF-alpha(Prepro)-(EA)3 (MRFPSIFTAVLFAASSALA-APVNTTTE-EGVSLEKR-EAEAEA (SEQ IDNO:11)), αF signal peptide(MRFPSIFTAVLFAASSALA-APVNTTTE-DETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINFTIASIAAKE-EGVSLEKR (SEQ ID NO:12)), KILM1 signal peptide(MTKPTQVLVRSVSILFFITLLHLVVALNDVAGPAETAPVSLLPR (SEQ ID NO:13)),repressible acid phosphatase (PHO1) signal peptide(MFSPILSLEIILALATLQSVFA (SEQ ID NO:14)), A. niger GOX signal peptide(MQTLLVSSLVVSLAAALPHYIR (SEQ ID NO:15)), Schwanniomyces occidentalisglucoamylase gene (GAM1) signal peptide (MIFLKLIKSIVIGLGLVSAIQA (SEQ IDNO:16)), human serum albumin (HSA) signal peptide with pro-sequence(MKWVTFISLLFLFSSAYSRGVFRR (SEQ ID NO:17)), human serum albumin (HSA)signal peptide without pro-sequence (MKWVTFISLLFLFSSAYS (SEQ ID NO:18)),ISN signal peptide (MALWMRLLPLLALLALWGPDPAAA (SEQ ID NO:19)), IFN signalpeptide (MKYTSYILAFQLCIVLGSLGCDLP (SEQ ID NO:20)), HGH signal peptide(MAADSQTPWLLTFSLLCLLWPQEPGA (SEQ ID NO:21)), phytohaemagglutinin (PHA)(MKKNRMMM MIWSVGVVWMLLLVGGSYG (SEQ ID NO:22)), Silkworm lysozyme(MQKLIIFALVVLCVGSEA (SEQ ID NO:23)), Human lysozyme (LYZ1)(MKALIVLGLVLLSVTVQG (SEQ ID NO:24)), activin receptor type-1(MVDGVMILPVLIMIALPSPS (SEQ ID NO:25)), activin type II receptor(MGAAAKLAFAVFLISCSSG (SEQ ID NO:26)), P. pastoris immunoglobulin bindingprotein (PpBiP) (MLSLKPSWLTLAALMYAMLLVVVPFAKPVRA (SEQ ID NO:27)), andhuman antibody 3D6 light chain leader (MDMRVPAQLLGLLLLWLPGAKC (SEQ IDNO:28)). See Hashimoto et al., Protein Engineering vol. 11 no. 2 pp.75-77, 1998; Oka et al., Biosci Biotechnol Biochem. 1999 November;63(11):1977-83; Gellissen et al., FEMS Yeast Research 5 (2005)1079-1096; Ma et al., Hepatology. 2005 December; 42(6):1355-63;Raemaekers et al., Eur J Biochem. 1999 Oct. 1; 265(1):394-403;Koganesawa et al., Protein Eng. (2001) 14 (9): 705-710; Daly et al.,Protein Expr Purif. 2006 April; 46(2):456-67; Damasceno et al., ApplMicrobiol Biotechnol (2007) 74:381-389; and Felgenhauer et al., NucleicAcids Res. 1990 Aug. 25; 18(16):4927, (each of which is incorporated byreference herein in its entirety).

The desired protein or complex may also be secreted into the culturemedia without being operably linked or fused to a secretion signal. Forexample, it has been demonstrated that some heterologous polypeptidesare secreted into the culture media when expressed in P. pastoris evenwithout being linked or fused to a secretion signal. Additionally, thedesired protein or multi-subunit complex may be purified from host cells(which, for example, may be preferable if the complex is poorlysecreted) using methods known in the art.

Media or cells comprising a desired protein or multi-subunit complex maybe recovered from the culture. Optionally, the secreted proteins may bepurified. For example, cells comprising a desired protein ormulti-subunit complex may be lysed using mechanical, chemical,enzymatic, and/or osmotic methods (e.g., freezing with liquid nitrogen,using a homogenizer, spheroplasting, sonication, agitation in thepresence of glass beads, using detergents, etc.). The desired protein ormulti-subunit complex may be concentrated, filtered, dialyzed, etc.,using methods known in the art. The desired protein or multi-subunitcomplex may be purified based on, for example, its molecular mass (e.g.,size exclusion chromatography), isoelectric point (e.g., isoelectricfocusing), electrophoretic mobility (e.g., gel electrophoresis),hydrophobic interaction chromatography (e.g., HPLC), charge (e.g., ionexchange chromatography), affinity (e.g., in the case of an antibody,binding to protein A, protein G, and/or an epitope to which the desiredantibody binds), and/or glycosylation state (e.g., detected by lectinbinding affinity). Multiple purification steps may be performed toobtain the desired level of purity. In an exemplary embodiment, thedesired protein or multi-subunit complex may be comprise animmunoglobulin constant domain and may be purified using protein A orprotein G affinity, size exclusion chromatography, and lack of bindingto lectin (to remove glycosylated forms). Optionally the A proteaseinhibitor, such as phenyl methyl sulfonyl fluoride (PMSF) may be addedto inhibit proteolytic degradation during purification.

Nucleic acids are “operably linked” when placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for asignal sequence is operably linked to DNA for a polypeptide if it isexpressed as a preprotein that participates in the secretion of thepolypeptide; a promoter or enhancer is operably linked to a codingsequence if it affects the transcription of the sequence. Generally,“operably linked” means that the DNA sequences being linked arecontiguous, and, in the case of a secretory leader, contiguous and inreading frame. However, enhancers do not have to be contiguous. Linkingmay be accomplished by ligation at convenient restriction sites oralternatively via a PCR/recombination method familiar to those skilledin the art (Gateway® Technology; Invitrogen, Carlsbad, Calif.). If suchsites do not exist, the synthetic oligonucleotide adapters or linkersmay be used in accordance with conventional practice. Desired nucleicacids (including nucleic acids comprising operably linked sequences) mayalso be produced by chemical synthesis.

Promoters are untranslated sequences located upstream (5′) to the startcodon of a structural gene (generally within about 100 to 1000 bp) thatcontrol the transcription and translation of particular nucleic acidsequences to which they are operably linked. Such promoters fall intoseveral classes: inducible, constitutive, and repressible promoters(that increase levels of transcription in response to absence of arepressor). Inducible promoters may initiate increased levels oftranscription from DNA under their control in response to some change inculture conditions, e.g., the presence or absence of a nutrient or achange in temperature.

The yeast promoter fragment may also serve as the site for homologousrecombination and integration of the expression vector into the samesite in the yeast genome; alternatively a selectable marker is used asthe site for homologous recombination. Pichia transformation isdescribed in Cregg et al. (1985) Mol. Cell. Biol. 5:3376-3385, which isincorporated by reference herein in its entirety.

Examples of suitable promoters from Pichia include the CUP1 (induced bythe level of copper in the medium), tetracycline inducible promoters,thiamine inducible promoters, AOX1 promoter (Cregg et al. (1989) Mol.Cell. Biol. 9:1316-1323); ICL1 promoter (Menendez et al. (2003) Yeast20(13):1097-108); glyceraldehyde-3-phosphate dehydrogenase promoter(GAP) (Waterham et al. (1997) Gene 186(1):37-44); and FLD1 promoter(Shen et al. (1998) Gene 216(1):93-102). The GAP promoter is a strongconstitutive promoter and the CUP1, AOX and FLD1 promoters areinducible. Each foregoing reference is incorporated by reference hereinin its entirety.

Other yeast promoters include ADH1, alcohol dehydrogenase II, GAL4,PHO3, PHO5, Pyk, and chimeric promoters derived therefrom. Additionally,non-yeast promoters may be used in the invention such as mammalian,insect, plant, reptile, amphibian, viral, and avian promoters. Mosttypically the promoter will comprise a mammalian promoter (potentiallyendogenous to the expressed genes) or will comprise a yeast or viralpromoter that provides for efficient transcription in yeast systems.

The polypeptides of interest may be produced recombinantly not onlydirectly, but also as a fusion polypeptide with a heterologouspolypeptide, e.g. a signal sequence or other polypeptide having aspecific cleavage site at the N-terminus of the mature protein orpolypeptide. In general, the signal sequence may be a component of thevector, or it may be a part of the polypeptide coding sequence that isinserted into the vector. The heterologous signal sequence selectedpreferably is one that is recognized and processed through one of thestandard pathways available within the host cell. The S. cerevisiaealpha factor pre-pro signal has proven effective in the secretion of avariety of recombinant proteins from P. pastoris. Other yeast signalsequences include the alpha mating factor signal sequence, the invertasesignal sequence, and signal sequences derived from other secreted yeastpolypeptides. Additionally, these signal peptide sequences may beengineered to provide for enhanced secretion in diploid yeast expressionsystems. Other secretion signals of interest also include mammaliansignal sequences, which may be heterologous to the protein beingsecreted, or may be a native sequence for the protein being secreted.Signal sequences include pre-peptide sequences, and in some instancesmay include propeptide sequences. Many such signal sequences are knownin the art, including the signal sequences found on immunoglobulinchains, e.g., K28 preprotoxin sequence, PHA-E, FACE, human MCP-1, humanserum albumin signal sequences, human Ig heavy chain, human Ig lightchain, and the like. For example, see Hashimoto et. al. Protein Eng11(2) 75 (1998); and Kobayashi et. al. Therapeutic Apheresis 2(4) 257(1998), each of which is incorporated by reference herein in itsentirety.

Transcription may be increased by inserting a transcriptional activatorsequence into the vector. These activators are cis-acting elements ofDNA, usually about from 10 to 300 bp, which act on a promoter toincrease its transcription. Transcriptional enhancers are relativelyorientation and position independent, having been found 5′ and 3′ to thetranscription unit, within an intron, as well as within the codingsequence itself. The enhancer may be spliced into the expression vectorat a position 5′ or 3′ to the coding sequence, but is preferably locatedat a site 5′ from the promoter.

Expression vectors used in eukaryotic host cells may also containsequences necessary for the termination of transcription and forstabilizing the mRNA. Such sequences are commonly available from 3′ tothe translation termination codon, in untranslated regions of eukaryoticor viral DNAs or cDNAs. These regions contain nucleotide segmentstranscribed as polyadenylated fragments in the untranslated portion ofthe mRNA.

Construction of suitable vectors containing one or more of theabove-listed components employs standard ligation techniques orPCR/recombination methods. Isolated plasmids or DNA fragments arecleaved, tailored, and re-ligated in the form desired to generate theplasmids required or via recombination methods. For analysis to confirmcorrect sequences in plasmids constructed, the ligation mixtures areused to transform host cells, and successful transformants selected byantibiotic resistance (e.g. ampicillin or Zeocin) where appropriate.Plasmids from the transformants are prepared, analyzed by restrictionendonuclease digestion and/or sequenced.

As an alternative to restriction and ligation of fragments,recombination methods based on att sites and recombination enzymes maybe used to insert DNA sequences into a vector. Such methods aredescribed, for example, by Landy (1989) Ann. Rev. Biochem. 58:913-949;and are known to those of skill in the art. Such methods utilizeintermolecular DNA recombination that is mediated by a mixture of lambdaand E. coli-encoded recombination proteins. Recombination occurs betweenspecific attachment (att) sites on the interacting DNA molecules. For adescription of att sites see Weisberg and Landy (1983) Site-SpecificRecombination in Phage Lambda, in Lambda II, Weisberg, ed. (Cold SpringHarbor, N.Y.: Cold Spring Harbor Press), pp. 211-250. The DNA segmentsflanking the recombination sites are switched, such that afterrecombination, the att sites are hybrid sequences comprised of sequencesdonated by each parental vector. The recombination can occur betweenDNAs of any topology. Each foregoing reference is incorporated byreference herein in its entirety.

Att sites may be introduced into a sequence of interest by ligating thesequence of interest into an appropriate vector; generating a PCRproduct containing att B sites through the use of specific primers;generating a cDNA library cloned into an appropriate vector containingatt sites; and the like.

Monocistronic and polycistronic genes. A monocistronic gene encodes anRNA that contains the genetic information to translate only a singleprotein. A polycistronic gene encodes an mRNA that contains the geneticinformation to translate more than one protein. The proteins encoded ina polycistronic gene may have the same or different sequences or acombination thereof. Dicistronic or bicistronic refers to apolycistronic gene that encodes two proteins. Polycistronic genesoptionally include one or more internal ribosome entry site (IRES)elements to facilitate cap-independent initiation of translation, whichmay be situated at a location that can drive translation of thedownstream protein coding region independently of the 5′-cap structurebound to the 5′ end of the mRNA molecule. Any known IRES sequence (e.g.,viral, eukaryotic, or artificial in origin) may be used. For example,the cricket paralysis virus IRES sequence in the intergenic region (IGR)may be used, as described in Thompson et al. (2001) PNAS 98:12972-12977.Optionally, IRES function may be potentiated by genetic alteration,e.g., by causing constitutive expression of eIF2 kinase GCN2 ordisrupting two initiator tRNA (met) genes disrupted (id).

Folding, as used herein, refers to the three-dimensional structure ofpolypeptides and proteins, where interactions between amino acidresidues act to stabilize the structure. While non-covalent interactionsare important in determining structure, usually the proteins of interestwill have intra- and/or intermolecular covalent disulfide bonds formedby two cysteine residues. For naturally occurring proteins andpolypeptides or derivatives and variants thereof, the proper folding istypically the arrangement that results in optimal biological activity,and can conveniently be monitored by assays for activity, e.g. ligandbinding, enzymatic activity, etc.

In some instances, for example where the desired product is of syntheticorigin, assays based on biological activity will be less meaningful. Theproper folding of such molecules may be determined on the basis ofphysical properties, energetic considerations, modeling studies, and thelike.

The expression host may be further modified by the introduction ofsequences encoding one or more enzymes that enhance folding anddisulfide bond formation, i.e. foldases, chaperonins, PDI, BIP,cyclophilin, etc. Such sequences may be constitutively or induciblyexpressed in the yeast host cell, using vectors, markers, etc. as knownin the art. Preferably the sequences, including transcriptionalregulatory elements sufficient for the desired pattern of expression,are stably integrated in the yeast genome through a targetedmethodology.

For example, the eukaryotic PD1 is not only an efficient catalyst ofprotein cysteine oxidation and disulfide bond isomerization, but alsoexhibits chaperone activity. Co-expression of PD1 can facilitate theproduction of active proteins having multiple disulfide bonds. Also ofinterest is the expression of BIP (immunoglobulin heavy chain bindingprotein); cyclophilin; and the like. In one embodiment of the invention,the multi-subunit complex may be expressed from a yeast strain producedby mating, wherein each of the haploid parental strains expresses adistinct folding enzyme, e.g. one strain may express BIP, and the otherstrain may express PD1 or combinations thereof.

The terms “desired protein” or “target protein” are used interchangeablyand refer generally to a heterologous multi-subunit protein such as anantibody (e.g., a humanized antibody) or a binding portion thereofdescribed herein.

The term “antibody” includes any polypeptide chain-containing molecularstructure with a specific shape that fits to and recognizes an epitope,where one or more non-covalent binding interactions stabilize thecomplex between the molecular structure and the epitope. The archetypalantibody molecule is the immunoglobulin, and all types ofimmunoglobulins, IgG, IgM, IgA, IgE, IgD, etc., from all sources, e.g.human, rodent, rabbit, cow, sheep, pig, dog, other mammals, chicken,other avians, etc., are considered to be “antibodies.” A preferredsource for producing antibodies useful as starting material according tothe invention is rabbits. Numerous antibody coding sequences have beendescribed; and others may be raised by methods well-known in the art.Examples thereof include chimeric antibodies, human antibodies and othernon-human mammalian antibodies, humanized antibodies, single chainantibodies such as scFvs, camelbodies, nanobodies, IgNAR (single-chainantibodies derived from sharks), small-modular immunopharmaceuticals(SMIPs), and antibody fragments such as Fabs, Fab′, F(ab′)₂ and thelike. See Streltsov V A, et al., Structure of a shark IgNAR antibodyvariable domain and modeling of an early-developmental isotype, ProteinSci. 2005 November; 14(11):2901-9. Epub 2005 Sep. 30; Greenberg A S, etal., A new antigen receptor gene family that undergoes rearrangement andextensive somatic diversification in sharks, Nature. 1995 Mar. 9;374(6518):168-73; Nuttall S D, et al., Isolation of the new antigenreceptor from wobbegong sharks, and use as a scaffold for the display ofprotein loop libraries, Mol Immunol. 2001 August; 38(4):313-26;Hamers-Casterman C, et al., Naturally occurring antibodies devoid oflight chains, Nature. 1993 Jun. 3; 363(6428):446-8; Gill D S, et al.,Biopharmaceutical drug discovery using novel protein scaffolds, CurrOpin Biotechnol. 2006 December; 17(6):653-8. Epub 2006 Oct. 19. Eachforegoing reference is incorporated by reference herein in its entirety.

For example, antibodies or antigen binding fragments may be produced bygenetic engineering. In this technique, as with other methods,antibody-producing cells are sensitized to the desired antigen orimmunogen. The messenger RNA isolated from antibody producing cells isused as a template to make cDNA using PCR amplification. A library ofvectors, each containing one heavy chain gene and one light chain generetaining the initial antigen specificity, is produced by insertion ofappropriate sections of the amplified immunoglobulin cDNA into theexpression vectors. A combinatorial library is constructed by combiningthe heavy chain gene library with the light chain gene library. Thisresults in a library of clones which co-express a heavy and light chain(resembling the Fab fragment or antigen binding fragment of an antibodymolecule). The vectors that carry these genes are co-transfected into ahost cell. When antibody gene synthesis is induced in the transfectedhost, the heavy and light chain proteins self-assemble to produce activeantibodies that can be detected by screening with the antigen orimmunogen.

Antibody coding sequences of interest include those encoded by nativesequences, as well as nucleic acids that, by virtue of the degeneracy ofthe genetic code, are not identical in sequence to the disclosed nucleicacids, and variants thereof. Variant polypeptides can include amino acid(aa) substitutions, additions or deletions. The amino acid substitutionscan be conservative amino acid substitutions or substitutions toeliminate non-essential amino acids, such as to alter a glycosylationsite, or to minimize misfolding by substitution or deletion of one ormore cysteine residues that are not necessary for function. Variants canbe designed so as to retain or have enhanced biological activity of aparticular region of the protein (e.g., a functional domain, catalyticamino acid residues, etc). Variants also include fragments of thepolypeptides disclosed herein, particularly biologically activefragments and/or fragments corresponding to functional domains.Techniques for in vitro mutagenesis of cloned genes are known. Alsoincluded in the subject invention are polypeptides that have beenmodified using ordinary molecular biological techniques so as to improvetheir resistance to proteolytic degradation or to optimize solubilityproperties or to render them more suitable as a therapeutic agent.

Chimeric antibodies may be made by recombinant means by combining thevariable light and heavy chain regions (V_(L) and V_(H)), obtained fromantibody producing cells of one species with the constant light andheavy chain regions from another. Typically chimeric antibodies utilizerodent or rabbit variable regions and human constant regions, in orderto produce an antibody with predominantly human domains. The productionof such chimeric antibodies is well known in the art, and may beachieved by standard means (as described, e.g., in U.S. Pat. No.5,624,659, incorporated herein by reference in its entirety). It isfurther contemplated that the human constant regions of chimericantibodies of the invention may be selected from IgG1, IgG2, IgG3, orIgG4 constant regions.

Humanized antibodies are engineered to contain even more human-likeimmunoglobulin domains, and incorporate only thecomplementarity-determining regions of the animal-derived antibody. Thisis accomplished by carefully examining the sequence of thehyper-variable loops of the variable regions of the monoclonal antibody,and fitting them to the structure of the human antibody chains. Althoughfacially complex, the process is straightforward in practice. See, e.g.,U.S. Pat. No. 6,187,287, incorporated fully herein by reference. Methodsof humanizing antibodies have been described previously in issued U.S.Pat. No. 7,935,340, the disclosure of which is incorporated herein byreference in its entirety. In some instances, a determination of whetheradditional rabbit framework residues are required to maintain activityis necessary. In some instances the humanized antibodies still requiressome critical rabbit framework residues to be retained to minimize lossof affinity or activity. In these cases, it is necessary to changesingle or multiple framework amino acids from human germline sequencesback to the original rabbit amino acids in order to have desiredactivity. These changes are determined experimentally to identify whichrabbit residues are necessary to preserve affinity and activity.

In addition to entire immunoglobulins (or their recombinantcounterparts), immunoglobulin fragments comprising the epitope bindingsite (e.g., Fab′, F(ab′)₂, or other fragments) may be synthesized.“Fragment,” or minimal immunoglobulins may be designed utilizingrecombinant immunoglobulin techniques. For instance “Fv” immunoglobulinsfor use in the present invention may be produced by synthesizing a fusedvariable light chain region and a variable heavy chain region.Combinations of antibodies are also of interest, e.g. diabodies, whichcomprise two distinct Fv specificities. In another embodiment of theinvention, SMIPs (small molecule immunopharmaceuticals), camelbodies,nanobodies, and IgNAR are encompassed by immunoglobulin fragments.

Immunoglobulins and fragments thereof may be modifiedpost-translationally, e.g. to add effector moieties such as chemicallinkers, detectable moieties, such as fluorescent dyes, enzymes, toxins,substrates, bioluminescent materials, radioactive materials,chemiluminescent moieties and the like, or specific binding moieties,such as streptavidin, avidin, or biotin, and the like may be utilized inthe methods and compositions of the present invention. Examples ofadditional effector molecules are provided infra.

Product-associated variant: a product other than the desired product(e.g., the desired multi-subunit complex) which is present in apreparation of the desired product and related to the desired product.Exemplary product-associated variants include truncated or elongatedpeptides, products having different glycosylation than the desiredglycosylation (e.g., if an aglycosylated product is desired then anyglycosylated product would be considered to be a product-associatedvariant), complexes having abnormal stoichiometry, improper assembly,abnormal disulfide linkages, abnormal or incomplete folding,aggregation, protease cleavage, or other abnormalities. Exemplaryproduct-associated variants may exhibit alterations in one or more ofmolecular mass (e.g., detected by size exclusion chromatography),isoelectric point (e.g., detected by isoelectric focusing),electrophoretic mobility (e.g., detected by gel electrophoresis),phosphorylation state (e.g., detected by mass spectrometry), charge tomass ratio (e.g., detected by mass spectrometry), mass or identity ofproteolytic fragments (e.g., detected by mass spectrometry or gelelectrophoresis), hydrophobicity (e.g., detected by HPLC), charge (e.g.,detected by ion exchange chromatography), affinity (e.g., in the case ofan antibody, detected by binding to protein A, protein G, and/or anepitope to which the desired antibody binds), and glycosylation state(e.g., detected by lectin binding affinity). Where the desired proteinis an antibody, the term product-associate variant may include aglyco-heavy variant and/or half antibody species (described below).

Exemplary product-associated variants include variant forms that containaberrant disulfide bonds. For example, most IgG1 antibody molecules arestabilized by a total of 16 intra-chain and inter-chain disulfidebridges, which stabilize the folding of the IgG domains in both heavyand light chains, while the inter-chain disulfide bridges stabilize theassociation between heavy and light chains. Other antibody typeslikewise contain characteristic stabilizing intra-chain and inter-chaindisulfide bonds. Further, some antibodies (including Ab-A and Ab-Bdisclosed herein) contain additional disulfide bonds referred to asnon-canonical disulfide bonds. Thus, aberrant inter-chain disulfidebonds may result in abnormal complex stoichiometry, due to the absenceof a stabilizing covalent linkage, and/or disulfide linkages toadditional subunits. Additionally, aberrant disulfide bonds (whetherinter-chain or intra-chain) may decrease structural stability of theantibody, which may result in decreased activity, decreased stability,increased propensity to form aggregates, and/or increasedimmunogenicity. Product-associated variants containing aberrantdisulfide bonds may be detected in a variety of ways, includingnon-reduced denaturing SDS-PAGE, capillary electrophoresis, cIEX, massspectrometry (optionally with chemical modification to produce a massshift in free cysteines), size exclusion chromatography, HPLC, changesin light scattering, and any other suitable methods known in the art.See, e.g., The Protein Protocols Handbook 2002, Part V, 581-583, DOI:10.1385/1-59259-169-8:581.

Half antibody, half-antibody species, or H1L1 refer to a protein complexthat includes a single heavy and single light antibody chain, but lacksa covalent linkage to a second heavy and light antibody chain. Two halfantibodies may remain non-covalently associated under some conditions,but may be separated under appropriate conditions (e.g., detergent,salt, or temperature) to facilitate their detection separate from fullH2L2 antibodies. Similarly, H2L1 refers to a protein complex thatincludes two heavy antibody chains and single light antibody chain, butlacks a covalent linkage to a second light antibody chain; thesecomplexes may also non-covalently associate with another light antibodychain (and likewise give similar behavior to a full antibody). Like fullantibodies, half antibody species and H2L1 species can dissociate underreducing conditions into individual heavy and light chains. Halfantibody species and H2L1 species can be detected on a non-reducedSDS-PAGE gel as a species migrating at a lower apparent molecular weightthan the full antibody, e.g., H1L1 migrates at approximately half theapparent molecular weight of the full antibody (e.g., about 75 kDa).

Glyco-heavy variant refers to a glycosylated product-associated variantsometimes present in antibody preparations and which contains at least apartial Fe sequence. The glyco-heavy variant is characterized bydecreased electrophoretic mobility observable by SDS-PAGE (relative to anormal heavy chain), lectin binding affinity, binding to an anti-Fcantibody, and apparent higher molecular weight of antibody complexescontaining the glyco-heavy variant as determined by size exclusionchromatography. See U.S. Provisional Application Ser. No. 61/525,307,filed Aug. 31, 2011 which is incorporated by reference herein in itsentirety.

The term “polyploid yeast that stably expresses or expresses a desiredsecreted heterologous polypeptide for prolonged time” refers to a yeastculture that secretes said polypeptide for at least several days to aweek, more preferably at least a month, still more preferably at least1-6 months, and even more preferably for more than a year at thresholdexpression levels, typically at least 50-500 mg/liter (after about 90hours in culture) and preferably substantially greater.

The term “polyploidal yeast culture that secretes desired amounts ofrecombinant polypeptide” refers to cultures that stably or for prolongedperiods secrete at least at least 50-500 mg/liter, and most preferably500-1000 mg/liter or more.

A polynucleotide sequence “corresponds” to a polypeptide sequence iftranslation of the polynucleotide sequence in accordance with thegenetic code yields the polypeptide sequence (i.e., the polynucleotidesequence “encodes” the polypeptide sequence), one polynucleotidesequence “corresponds” to another polynucleotide sequence if the twosequences encode the same polypeptide sequence.

A “heterologous” region or domain of a DNA construct is an identifiablesegment of DNA within a larger DNA molecule that is not found inassociation with the larger molecule in nature. Thus, when theheterologous region encodes a mammalian gene, the gene will usually beflanked by DNA that does not flank the mammalian genomic DNA in thegenome of the source organism. Another example of a heterologous regionis a construct where the coding sequence itself is not found in nature(e.g., a cDNA where the genomic coding sequence contains introns, orsynthetic sequences having codons different than the native gene).Allelic variations or naturally-occurring mutational events do not giverise to a heterologous region of DNA as defined herein.

A “coding sequence” is an in-frame sequence of codons that (in view ofthe genetic code) correspond to or encode a protein or peptide sequence.Two coding sequences correspond to each other if the sequences or theircomplementary sequences encode the same amino acid sequences. A codingsequence in association with appropriate regulatory sequences may betranscribed and translated into a polypeptide. A polyadenylation signaland transcription termination sequence will usually be located 3′ to thecoding sequence. A “promoter sequence” is a DNA regulatory regioncapable of binding RNA polymerase in a cell and initiating transcriptionof a downstream (3′ direction) coding sequence. Promoter sequencestypically contain additional sites for binding of regulatory molecules(e.g., transcription factors) which affect the transcription of thecoding sequence. A coding sequence is “under the control” of thepromoter sequence or “operatively linked” to the promoter when RNApolymerase binds the promoter sequence in a cell and transcribes thecoding sequence into mRNA, which is then in turn translated into theprotein encoded by the coding sequence.

Vectors are used to introduce a foreign substance, such as DNA, RNA orprotein, into an organism or host cell. Typical vectors includerecombinant viruses (for polynucleotides) and liposomes (forpolypeptides). A “DNA vector” is a replicon, such as plasmid, phage orcosmid, to which another polynucleotide segment may be attached so as tobring about the replication of the attached segment. An “expressionvector” is a DNA vector which contains regulatory sequences which willdirect polypeptide synthesis by an appropriate host cell. This usuallymeans a promoter to bind RNA polymerase and initiate transcription ofmRNA, as well as ribosome binding sites and initiation signals to directtranslation of the mRNA into a polypeptide(s). Incorporation of apolynucleotide sequence into an expression vector at the proper site andin correct reading frame, followed by transformation of an appropriatehost cell by the vector, enables the production of a polypeptide encodedby said polynucleotide sequence.

“Amplification” of polynucleotide sequences is the in vitro productionof multiple copies of a particular nucleic acid sequence. The amplifiedsequence is usually in the form of DNA. A variety of techniques forcarrying out such amplification are described in the following reviewarticles, each of which is incorporated by reference herein in itsentirety: Van Brunt 1990, Bio/Technol., 8(4):291-294; and Gill andGhaemi, Nucleosides Nucleotides Nucleic Acids. 2008 March; 27(3):224-43.Polymerase chain reaction or PCR is a prototype of nucleic acidamplification, and use of PCR herein should be considered exemplary ofother suitable amplification techniques.

The general structure of antibodies in most vertebrates (includingmammals) is now well understood (Edelman, G. M., Ann. N.Y. Acad. Sci.,190: 5 (1971)). Conventional antibodies consist of two identical lightpolypeptide chains of molecular weight approximately 23,000 daltons (the“light chain”), and two identical heavy chains of molecular weight53,000-70,000 (the “heavy chain”). The four chains are joined bydisulfide bonds in a “Y” configuration wherein the light chains bracketthe heavy chains starting at the mouth of the “Y” configuration. The“branch” portion of the “Y” configuration is designated the F_(ab)region; the stem portion of the “Y” configuration is designated theF_(C) region. The amino acid sequence orientation runs from theN-terminal end at the top of the “Y” configuration to the C-terminal endat the bottom of each chain. The N-terminal end possesses the variableregion having specificity for the antigen that elicited it, and isapproximately 100 amino acids in length, there being slight variationsbetween light and heavy chain and from antibody to antibody.

The variable region is linked in each chain to a constant region thatextends the remaining length of the chain and that within a particularclass of antibody does not vary with the specificity of the antibody(i.e., the antigen eliciting it). There are five known major classes ofconstant regions that determine the class of the immunoglobulin molecule(IgG, IgM, IgA, IgD, and IgE corresponding to gamma, mu, alpha, delta,and epsilon heavy chain constant regions). The constant region or classdetermines subsequent effector function of the antibody, includingactivation of complement (Kabat, E. A., Structural Concepts inImmunology and Immunochemistry, 2nd Ed., p. 413-436, Holt, Rinehart,Winston (1976)), and other cellular responses (Andrews, D. W., et al.,Clinical Immunobiology, pp 1-18, W. B. Sanders (1980); Kohl, S., et al.,Immunology, 48: 187 (1983)); while the variable region determines theantigen with which it will react. Light chains are classified as eitherkappa or lambda. Each heavy chain class can be paired with either kappaor lambda light chain. The light and heavy chains are covalently bondedto each other, and the “tail” portions of the two heavy chains arebonded to each other by covalent disulfide linkages when theimmunoglobulins are generated either by hybridomas or by B cells.

The expression “variable region” or “VR” refers to the domains withineach pair of light and heavy chains in an antibody that are involveddirectly in binding the antibody to the antigen. Each heavy chain has atone end a variable domain (V_(H)) followed by a number of constantdomains. Each light chain has a variable domain (V_(L)) at one end and aconstant domain at its other end; the constant domain of the light chainis aligned with the first constant domain of the heavy chain, and thelight chain variable domain is aligned with the variable domain of theheavy chain.

The expressions “complementarity determining region,” “hypervariableregion,” or “CDR” refer to one or more of the hyper-variable orcomplementarity determining regions (CDRs) found in the variable regionsof light or heavy chains of an antibody (See Kabat, E. A. et al.,Sequences of Proteins of Immunological Interest, National Institutes ofHealth, Bethesda, Md., (1987)). These expressions include thehypervariable regions as defined by Kabat et al. (“Sequences of Proteinsof Immunological Interest,” Kabat E., et al., US Dept. of Health andHuman Services, 1983) or the hypervariable loops in 3-dimensionalstructures of antibodies (Chothia and Lesk, J Mol. Biol. 196 901-917(1987)). The CDRs in each chain are held in close proximity by frameworkregions and, with the CDRs from the other chain, contribute to theformation of the antigen binding site. Within the CDRs there are selectamino acids that have been described as the selectivity determiningregions (SDRs) which represent the critical contact residues used by theCDR in the antibody-antigen interaction (Kashmiri, S., Methods, 36:25-34(2005)).

The expressions “framework region” or “FR” refer to one or more of theframework regions within the variable regions of the light and heavychains of an antibody (See Kabat, E. A. et al., Sequences of Proteins ofImmunological Interest, National Institutes of Health, Bethesda, Md.,(1987)). These expressions include those amino acid sequence regionsinterposed between the CDRs within the variable regions of the light andheavy chains of an antibody.

The expression “stable copy number” refers to a host cell thatsubstantially maintains the number of copies of a gene (such as anantibody chain gene) over a prolonged period of time (such as at least aday, at least a week, or at least a month, or more) or over a prolongednumber of generations of propagation (e.g., at least 30, 40, 50, 75,100, 200, 500, or 1000 generations, or more). For example, at a giventime point or number of generations, at least 50%, and preferably atleast 70%, 75%, 85%, 90%, 95%, or more of cells in the culture maymaintain the same number of copies of the gene as in the starting cell.In a preferred embodiment, the host cell contains a stable copy numberof the gene encoding the desired protein or encoding each subunit of thedesired multi-subunit complex (e.g., antibody).

The expression “stably expresses” refers to a host cell that maintainssimilar levels of expression of a gene or protein (such as an antibody)over a prolonged period of time (such as at least a day, at least aweek, or at least a month, or more) or over a prolonged number ofgenerations of propagation (e.g., at least 30, 40, 50, 75, 100, 200,500, or 1000 generations, or more). For example, at a given time pointor number of generations, the rate of production or yield of the gene orprotein may be at least 50%, and preferably at least 70%, 75%, 85%, 90%,95%, or more of the initial rate of production. In a preferredembodiment, the host cell stably expresses the desired protein ormulti-subunit complex (e.g., antibody).

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the subject invention, and are not intended to limit thescope of what is regarded as the invention. Efforts have been made toensure accuracy with respect to the numbers used (e.g. amounts,temperature, concentrations, etc.) but some experimental errors anddeviations should be allowed for. Unless otherwise indicated, parts areparts by weight, molecular weight is average molecular weight,temperature is in degrees centigrade; and pressure is at or nearatmospheric.

The above disclosure generally describes the present invention. Allreferences disclosed herein are expressly incorporated by reference. Amore complete understanding can be obtained by reference to thefollowing specific examples which are provided herein for purposes ofillustration only, and are not intended to limit the scope of theinvention.

Example 1

Examples 1 through 10 show the applicability of the method to theproduction of four different humanized monoclonal antibodies. Eachantibody is produced in Pichia pastoris using theglyceraldehyde-3-phosphate (GAP) promoter system.

We found a difference in titers between aerobic and hypoxic cultures ofantibody Mab2. Restricting the oxygen availability to the culture byreducing the agitation rate in the fermentor resulted in a significantincrease in product formation. This was our first confirmation thathypoxic conditions, when applied to the production of full lengthantibodies, results in a significant increase in product formation forfully assembled, appropriately disulfide bonded humanized monoclonalantibodies. See FIG. 37.

Example 2

Three different strains of antibody Mab1, each with differing copynumbers, are grown in 20 liter fermentors using RQ control strategy(modulation of agitation using feedback control that modulates agitatorspeed to maintain RQ at the desired level (in this case a value of 1.12)to promote mixed metabolism of hypoxic conditions in a controlled mannerso as to ensure that ethanol concentrations do not reach toxic levels.In each case, the robust nature of the feedback control mechanism allowsmixed metabolism without accumulation of toxic levels of ethanol(typically greater than 20 g/L). (See FIGS. 1-5)

Example 3

Mab1 is cultured under hypoxic condition using the RQ control strategy,at three different control set-points for RQ. In this case, increasingthe RQ set-point increases the level of ethanol accumulation, reducesthe accumulation of cells, but does not have a significant impact on theoverall product accumulation. This shows the utility of the RQ method atset-points ranging from 1.09 to 1.35. (See FIGS. 6-10).

Example 4

We compared, for Mab1, the effect of hypoxic growth, as attained by RQcontrol, against the same process under aerobic conditions. The aerobicprocess results in lower ethanol production (as expected), and markedlylower product formation. (See FIGS. 11-16).

Example 5

The RQ control strategy was implemented on fermentations of Mab2 thathad production strains varying in the number of copies of each heavy andlight chain. This study shows the robust nature of the RQ strategy incontrolling the accumulation of ethanol while providing a hypoxicenvironment for mixed metabolism. (See FIGS. 17-21).

Example 6

The RQ control strategy was implemented on fermentations of Mab3, thathad production strains varying in the number of copies of each heavy andlight chain. This study shows the robust nature of the RQ strategy incontrolling the accumulation of ethanol while providing a hypoxicenvironment for mixed metabolism. (See FIGS. 22-26)

Example 7

Strains of Mab3, containing varying copies of Heavy Chain and of LightChain is grown using the RQ control strategy but incorporating varyingglucose feed rates. Again, the RQ strategy allows for effective controlof ethanol levels, resulting in very similar product accumulation rates.This provides further evidence of the robustness of the RQ strategy to avariety of fermentation conditions. (See FIGS. 27-31).

Example 8

The RQ strategy is demonstrated for MAb4, which binds to the same targetas Mab1, but has a different sequence in its CDR than Mab1. We comparedtwo different rates of glucose feed. Once again the strategy allowed fora stable ethanol concentration and similar antibody accumulation rates.(See FIGS. 32-36).

Example 9

This example describes the fermentation process for the production ofantibodies or antigen-binding Fermentation Media.

Inoculum Medium is described below in Table 1.

TABLE 1 Inoculum Medium Component* Final concentration Yeast extract 30g/l KH2PO4 27.2 g/l Glycerol or Glucose 20 g/l Yeast nitrogen base w/oamino acids 13.4 g/l Biotin 0.4 mg/l *Keeping the same molarity, anychemical (X nH₂O; n ≥ 0) can be replaced by another chemical containingthe same activated ingredient but various amount of water (X kH₂O; k ≠n).

Seed Fermentation Medium

Medium is described below in Table 2.

Composition Seed Fermentation Medium

Component* Final concentration Sodium citrate dihydrate 10.0 g/lMgSO4—7H2O 3.7 g/l NH4H2PO4 36.4 g/l K2HPO4 12.8 g/l K2SO4 18.2 g/lGlycerol, anhydrous 40.0 g/l Yeast extract 30.0 g/l Antifoam 204 0.5ml/l Trace mineral solution (PTM1) 4.35 ml/l *Keeping the same molarity,any chemical (X nH₂O; n ≥ 0) can be replaced by another chemicalcontaining the same activated ingredient but various amount of water (XkH₂O; k ≠ n).

Trace Mineral Solution is described below in Table 3.

TABLE 3 Trace Mineral Solution (PTM1) Component* Final concentrationZnCl21 or Zinc Sulphate Heptahydrate2 20 g/l1 or 35 g/l2 FeSO4—7H2O 65g/l 95-98% H2SO4 5 ml/l NaI 0.08 g/l MnSO4—2H2O 3 g/l Na2MoO4—2H2O 0.2g/l H3BO3 0.02 g/l CoCl2 0.5 g/l CuSO4—5H2O 6 g/l Biotin 0.2 g/l*Keeping the same molarity, any chemical (X nH₂O; n ≥ 0) can be replacedby another chemical containing the same activated ingredient but variousamount of water (X kH₂O; k ≠ n).

When all components are completely dissolved in DI water, filtersterilize through a sterile 0.2 μm filter.

Production Culture Batch Medium is described below in Table 4

TABLE 4 Production Culture Batch Medium Component* Final concentrationSodium citrate dihydrate 10.0 g/l MgSO4—7H2O 3.7 g/l NH4H2PO4 35.6 g/lK2HPO4 12.8 g/l K2SO4 18.2 g/l Glycerol, anhydrous 40.0 g/l Yeastextract 30.0 g/l Antifoam 204 1.6 ml/l *Keeping the same molarity, anychemical (X nH2O; n ≥ 0) can be replaced by another chemical containingthe same activated ingredient but various amount of water (X kH2O; k ≠n).

The above is sterilized by autoclaving at 121° C. for a minimum of 20minutes. After sterilization and cooling, 4.35 ml/l of trace mineralsolution (PTM1) is added to the Production Culture Batch Medium. Priorto inoculation of the fermentor, Production Culture Batch Mediumcontaining 4.35 ml/l of PTM1 should be adjusted to pH 6.0 with 24-30%NH₄OH. The above values should be based on the total fermentationstarting volume, including both medium and inoculum culture.

Glucose/Yeast Extract Feed Solution is described below in Table 5.

Component* Final concentration Dextrose, anhydrous 500 g/l Yeast extract50 g/l MgSO4—7H2O 3 g/l Antifoam 204 0.1 ml/l Sodium citrate dihydrate1.66 g/l PTM1 12 ml/l *Keeping the same molarity, any chemical (X nH₂O;n ≥ 0) can be replaced by another chemical containing the same activatedingredient but various amount of water (X kH₂O; k ≠ n).

Ethanol bolus composition is described below in Table 6.

Component* Final concentration Ethanol, 200 Proof 11 g/l *Optionally amore dilute solution of ethanol can be used to achieve the same finalconcentration.

Fermentation Process

The fermentation process for the production of antibodies orantigen-binding fragments is accomplished by yeast, such as P. pastoris.The fermentation is initiated from the thawing of a frozen vial of aworking cell bank. The thawed cells are then propagated in shake flasks.The culture from the shake flask is then used in the Inoculum Step,followed by a fed-batch process for the production of antibody.Optionally, the inoculum can be used to propagate cells in a seed batchfermentation, which can then be used to inoculate the productionfermentor.

1. Inoculum Step

Thawed cells of the working cell bank are transferred to a baffled shakeflask (1 to 4 baffles) that contains 8-20% of the working volumecapacity of the flask Inoculum Medium. Thawed working cell bank is addedat 0.1-1.0% of the volume of inoculum medium to the shake flask. Theinoculum culture is incubated at 29-31° C. at an agitation speed of220-260 rpm. The seed culture is harvested once reaching a cell densitycorrelated to the absorbance at 600 nm (OD₆₀₀) of 15-30 (optimally20-30). The culturing time is usually 20-26 hours (optimally 23-25hours).

2. Seed Fermentation Batch Fermentation (Optional)

Fermentor is inoculated with inoculum from “Inoculum Step”

Inoculum=0.3% of seed fermentor medium volume

Temp: 30° C.

% DO: 30%

pH: 6.0

Agitation: Cascade Strategy from 100-490 RPM

Airflow: 1 vvm ((volume of air/volume of starting fermentormedia)/minute)

Pressure: 0.2 bar

Oxygen supplementation will occur when maximum agitation is reached witha corresponding decrease in airflow to maintain a constant vvm, tomaintain the desired % DO set point of 30%

Continue Monitoring for DO Spike.

When a DO Spike has occurred which is indicated by a decrease inAgitation and an increase in DO, denoting that the carbon source(glycerol or glucose) has been completely utilized and the measuredoptical density, OD₆₀₀, is greater than 20, transfer a volume of theseed batch fermentation or inoculum culture which is equal to 1.0-10% ofthe Production fermentor Starting Batch Volume.

3. Batch Culture Phase

The batch culture is initiated by inoculation of the fermentor with theseed culture and ended with the depletion of glycerol. The fermentorcontains prepared Production Culture Batch medium at 30-40% of maximumworking volume. The seed culture is used to create a 1-10% inoculumwithin the fermentor. The initial engineering parameters are set asfollows:

-   -   Temperature: 27-29° C.;    -   Agitation (P/V): 2-16 KW/m³    -   Headspace Pressure: 0.7-0.9 Bar    -   Air flow: 0.9-1.4 VVM (volumes of air per volume of culture per        minute, based on starting volume)    -   DO: no control    -   pH: 5.9 to 6.1, controlled by 24-30% NH₄OH

The starting agitation speed and airflow are kept constant during theBatch Culture Phase in order to meet the initial power per volume (Ply)and volume per volume per minute (VVM) requirements. Batch Culture Phaseis ended by starting feed when glycerol is depleted. The depletion ofglycerol is indicated by the dissolved oxygen (DO) value spike. The DOspike is defined as when the value of the DO increases by greater than30% within a few minutes. Batch Culture Phase usually lasts 10-15 hours(optimally 11-13 hours).

4. Ethanol Bolus Addition (Optional)

Upon observation of the DO spike as mentioned above, 8-16 g/l ofEthanol, 200 Proof as a bolus is added into the fermentor. This usuallyoccurs within 12-14 hours of Batch Culture Phase.

5. Fed-Batch Culture Phase

Feed to the fermentor with Glucose/Yeast Extract Feed Solution isinitiated after the DO spike and after Ethanol Bolus Addition, around12-14 hours within Batch Culture Phase and continues to the end of thefermentation. The rate of Glucose/Yeast Extract Feed Solution feed isset to allow for 6-11 g of glucose/l of starting volume per hour. Thestart of Glucose/Yeast Extract Feed Solution begins Fed-batch CulturePhase.

6. RQ Control Start

Respiratory Quotient (RQ) Control begins 8 hours after Fed-batch CulturePhase start. The initial RQ set points are in the range of 1.09 to 1.35.Agitation is used to control the RQ. Agitation is cascaded off of the RQcontrol set point. RQ control starts at approximately 20-22 hours fromthe onset of Batch Culture Phase and continues to the end of thefermentation. The duration of RQ control lasts approximately 60 to 90hours.

The agitation is adjusted in order to maintain a set level of RQ. The RQControl strategy is detailed as follows:

-   -   RQ Hi Control set point: 1.35    -   RQ Low Control set point: 1.08    -   Maximum agitator set point: 255-950 rpm    -   Minimum agitator set point: 150-300 rpm    -   Agitator step change (change at each Wait Time interval): 3-25        rpm    -   Wait Time (time between evaluations): 3-10 minutes

Ethanol/RQ Control Strategy

This strategy has been incorporated to ensure that the ethanolconcentration does not exceed a maximum value that can be toxic to thecells, and does not exceed a minimum value that could reduce productexpression.

Example 10

FIG. 38 show SDS-PAGE gels of Mab1 produced under both hypoxic andaerobic conditions. For the non-reduced gel, in addition to the mainband at 150 kD, additional bands below the main band indicate productheterogeneity with respect to the level of interchain disulfidebridging. These gels show that the level of heterogeneity is reduced bythe use of hypoxic conditions. The increased homogeneity of the fulllength, completely cross-linked product indicates increased purity,i.e., increased desired product relative to other proteins present.

FIG. 38 also shows the reduced SDS-PAGE gel for the same samples. Inthis case expected bands at 25 kD and 50 kD represent the heavy andlight chains of the antibody. The additional bands, particularly the oneabove the Heavy chain at approximately 55 kD represent the present ofvariant species of the antibody. These gels show a dramatic reduction inthe presence of this variant when the cells are grown under hypoxicconditions, as compared with the aerobic culture.

Example 11

This example tests the effect of a temperature shift on yield and purityof antibodies expressed from P. pastoris. Antibody yield was increasedby up to about 30% by an upward temperature shift effected duringculture. Additionally, purity was increased by the temperature shift, asindicated by a decrease in the abundance of product-associated variantsand aberrant complexes.

Methods

Ab-A was expressed from a P. pastoris strain containing 4 integratedcopies of the heavy chain gene and 3 integrated copies of the lightchain gene (SEQ ID NOS: 1 and 2, respectively). An inoculum was expandedusing a medium comprised of the following nutrients (% w/v): yeastextract 3%, glycerol 2%, YNB 1.34%, Biotin 0.004% and 27.2 g/l potassiumphosphate monobasic. To generate the inoculum for the fermenters, thecells were grown for approximately 24-28 hours in a shaking incubator at30° C. and 300 rpm.

The Ab-A sequences are as follows:

Ab-A heavy chain polynucleotide sequence: (SEQ ID NO: 1)gaggtgcagcttgtggagtctgggggaggcttggtccagcctggggggtccctgagactctcctgtgcagtctctggaatcgacctcagtggctactacatgaactgggtccgtcaggctccagggaaggggctggagtgggtcggagtcattggtattaatggtgccacatactacgcgagagggcgaaaggccgattcaccatctccagagacaattccaagaccacggtgtatcttcaaatgaacagcctgagagctgaggacactgctgtgtatttctgtgctagaggggacatctggggccaagggaccctcgtcaccgtctcgagcgcctccaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacgcgagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtatcctcttccccccaaaacccaaggacaccctcatgatctcccggacccagaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacgccagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtaccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttatcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtatctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctct ccctgtctccgggtaaatgaAb-A light chain polynucleotide sequence: (SEQ ID NO: 2)caagtgctgacccagtctccatcctccagtctgcatctgtaggagacagagtcaccatcaattgccaggccagtcagagtgtttatcataacacctacctggcctggtatcagcagaaaccagggaaagttcctaagcaactgatctatgatgcatccactctggcatctggggtcccatctcgtttcagtggcagtggatctgggacagattttcactctcaccatcagcagcctgcagcctgaagatgttgcaacttattactgtctgggcagttatgattgtactaatggtgattgttttgttttcggcggaggaaccaaggtggaaatcaaacgtacggtggctgcaccatctgtcttcatatcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccaccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggag agtgttag 

A 10% inoculum was then added to Applikon 17L working volume vesselscontaining 6 L sterile growth medium. The growth medium was comprised ofthe following nutrients: potassium sulfate 18.2 g/L, ammonium phosphatemonobasic 35.6 g/L, potassium phosphate dibasic 12.8 g/L, magnesiumsulfate heptahydrate 3.72 g/L, sodium citrate dihydrate 10 g/L, glycerol40 g/L, yeast extract 30 g/L, PTM1 trace metals 4.35 mL/L, and antifoam204 1.67 mL/L. The PTM1 trace metal solution was comprised of thefollowing components: cupric sulfate pentahydrate 6 g/L, sodium iodide0.08 g/L, manganese sulfate hydrate 3 g/L, sodium molybdate dihydrate0.2 g/L, boric acid 0.02 g/L, cobalt chloride 0.5 g/L, zinc chloride 20g/L, ferrous sulfate heptahydrate 65 g/L, biotin 0.2 g/L, and sulfuricacid 5 mL/L. The bioreactor process control parameters were set asfollows: Agitation 950 rpm, airflow 1.35 standard liter per minute,temperature 28° C. and pH was controlled (at 6) using ammoniumhydroxide. No oxygen supplementation was provided.

Fermentation cultures were grown for approximately 12 to 16 hours at 28°C. until the initial glycerol was consumed, which was detected by arapid increase in the concentration of dissolved oxygen, referred to asthe DO spike. Immediately after the DO spike was detected, a bolus of 11grams of 100% ethanol per liter of culture was added to the reactor toattain a final concentration of about 1.1% ethanol (w/v). Thefermentation cultures were allowed to equilibrate for 20 minutes. Feedaddition was then initiated at a constant rate of 11 g glucose/L/hr forthe duration of the fermentation. Approximately 8 hrs after the feedaddition was initiated, RQ control was initiated using agitation feedback control with a minimum agitation speed of 500 rpm and a maximumagitation speed of 950 rpm thereby maintaining the RQ set point of 1.12for the remainder of the fermentation. The feed was comprised of thefollowing components: yeast extract 50 g/L, dextrose anhydrous 500 g/L,magnesium sulfate heptahydrate 3 g/L, and PTM1 trace metals 12 mL/L.Optionally, sodium citrate dihydrate (1.66 g/L) was also added to thefeed. Feed pH was 6.0.

Five minutes after feed initiation, the culture temperature was rapidlyshifted to one of five different temperatures (25° C., 29.5° C., 31° C.,32.5° C., and 34° C.). Additionally, a control culture was maintained at28° C., i.e. without temperature shift. The total fermentation time wasapproximately 86-87 hours.

Samples were collected from each culture throughout the fermentation andwhole broth titers were determined and were plotted in arbitrary unitswhich are consistent among the figures in this application.Additionally, at the end of the run, antibody purity was determined(after Protein A purification) by size-exclusion chromatography(SE-HPLC) performed on reduced and non-reduced samples using an Agilent(Santa Clara, Calif.) 1200 Series HPLC with UV detection instrument. Forsample separation, a TSKgel GS3000SW×1 7.8×300 mM column connected witha TSKgel Guard SW×1 6×40 mM from Tosoh Bioscience (King of Prussia, Pa.)was used. A 100 mM sodium phosphate, 200 mM sodium chloride pH 6.5 wasused as mobile phase with a flow rate of 0.5 mL/min in isocratic modeand absorbance at UV 215 nm was monitored. Before injection of samplesthe column was equilibrated until a stable baseline was achieved.Samples were diluted to a concentration of 1 mg/mL using mobile phaseand a 30 μL volume was injected. To monitor column performance, BioRad(Hercules, Calif.) gel filtration standards were used.

Results

P. pastoris engineered to express Ab-A was grown in cultures maintainedat 28° C. during an initial growth phase with glycerol as a carbonsource. After exhaustion of the glycerol, a continuous glucose feed wasinitiated and the culture temperature was rapidly shifted upward ordownward to a new set-point temperature between 25° C. and 34° C. whichwas maintained for the duration of the culture. One culture wasmaintained at 28° C. as a control.

To monitor antibody production, culture media (into which the antibodywas secreted due to inclusion of a secretion signal) was periodicallysampled up to the final time-point of 86-87 hours. Whole broth antibodytiter (arbitrary units) was determined and is shown graphically for eachculture temperature in FIG. 40. As depicted, the highest final titer wasachieved for the culture maintained at 31° C. A slightly higher titerwas initially obtained for the culture maintained at 32.5° C., howeverthe whole broth titer leveled off and began to decrease between 65-70hours, and the final titer was lower than was observed for thenon-shifted culture. The second-highest final titer was observed withthe culture maintained at 29.5° C. The culture shifted up to 34° C. andthe culture shifted downward to 25° C. both produced titers lower thanthe culture maintained at 28° C.

Additionally, antibody purity at the end of culture (i.e., at 86-87hours) was determined. Specifically, protein-A purified antibody fromeach culture was assessed by exclusion chromatography (SEC) and by gelelectrophoresis with Coomassie staining (FIGS. 42-47), which wereconducted for both reduced and non-reduced samples. SEC analysis ofnon-reduced samples detected relative abundance of complexes havingaberrant stoichiometry, including a 75 kDA “half antibody” speciescontaining a single heavy chain and a single light chain, and a “HHL”complex containing two heavy chains and a single light chain. SECanalysis of reduced samples detected relative abundance of intact heavyand light chains as well as aberrant subunits having an elution time ofapproximately 9.8, 10.15, and 10.8 minutes (which are thought tocorrespond to glycosylated forms of the antibody heavy chain).

The SEC results are summarized in FIG. 41A-B. The non-reduced SECanalysis demonstrated that a similar proportion of the total protein wascontained in the main antibody peak for unshifted cultures (i.e.,maintained at 28° C.) and cultures shifted to 29.5° C. and 31° C., withall three conditions maintaining 88-89% of total protein within the fullantibody peak (FIG. 41A). Thus, with respect to misassembled complexes,the upward shift to 29.5° C. and 31° C. did not adversely affect purity.Further, the reduced SEC analysis demonstrated that compared to theunshifted culture, the proportion of total protein contained in theheavy and light chain peaks remained similar for the culture shifted to29.5° C. and was increased by about 4% for higher temperature shifts.

Based thereon, it is concluded that upward temperature shifts to 29.5°C. or 31° C. (i.e., by 1.5° C. to 3° C.) increased final antibody yield.Additionally, purity was increased in the culture shifted to 31° C.,while purity was not adversely affected for the culture shifted to 29.5°C.

Example 12

This example tests the effect of a temperature shift on yield and purityof antibodies expressed from P. pastoris. Two different strains weretested, a lower- and higher-producing strain. For the higher-producingstrain, antibody yield was increased by about 28% by an upwardtemperature shift effected during culture. Based thereon it is concludedthat production from an already highly optimized strain wassubstantially benefited by the temperature shift. Additionally, puritywas increased by the temperature shift, as indicated by a decrease inthe abundance of product-associated variants in most instances.

Methods

Ab-B was expressed from a P. pastoris strain containing 3 or 4integrated copies of the heavy chain gene and 3 integrated copies of thelight chain gene (SEQ ID NOS: 3 and 4, respectively. An inoculum wasexpanded using a medium comprised of the following nutrients (% w/v):yeast extract 3%, glycerol 2%, YNB 1.34%, Biotin 0.004% and 27.2 g/lpotassium phosphate monobasic. To generate the inoculum for thefermenters, the cells were grown for approximately 24-28 hours in ashaking incubator at 30° C. and 300 rpm.

The Ab-B sequences are as follows:

Ab-B heavy chain polynucleotide sequence: (SEQ ID NO: 3)gaggtgcagctggtggagtctgggggaggcttggtccagcctggggggtccctgagactctcctgtgcagcctctggattctccctcagtaactactacgtgacctgggtccgtcaggctccagggaaggggctggagtgggtcggcatcatctatggtagtgatgaaaccgcctacgctacctccgctataggccgattcaccatctccagagacaattccaagaacaccctgtatcttcaaatgaacagcctgagagctgaggacactgctgtgtattactgtgctagagatgatagtagtgactgggatgcaaagttcaacttgtggggccaagggaccctcgtcaccgtctcgagcgcctccaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacgccagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggaccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtaccgggtaaaAb-B light chain polynucleotide sequence: (SEQ ID NO: 4)gctatccagatgacccagtctccttcctccctgtctgcatctgtaggagacagagtcaccatcacttgccaggccagtcagagcattaacaatgagttatcctggtatcagcagaaaccagggaaagcccctaagctcctgatctatagggcatccactctggcatctggggtcccatcaaggttcagcggcagtggatctgggacagacttcactctcaccatcagcagcctgcagcctgatgattttgcaacttattactgccaacagggttatagtagaggaacattgataatgcatcggcggagggaccaaggtggaaatcaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgagaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccagacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt

A 10% inoculum was then added to Applikon 17L working volume vesselscontaining 6 L sterile growth medium. The growth medium was comprised ofthe following nutrients: potassium sulfate 18.2 g/L, ammonium phosphatemonobasic 35.6 g/L, potassium phosphate dibasic 12.8 g/L, magnesiumsulfate heptahydrate 3.72 g/L, sodium citrate dihydrate 10 g/L, glycerol40 g/L, yeast extract 30 g/L, PTM1 trace metals 4.35 mL/L, and antifoam204 1.67 mL/L. The PTM1 trace metal solution was comprised of thefollowing components: cupric sulfate pentahydrate 6 g/L, sodium iodide0.08 g/L, manganese sulfate hydrate 3 g/L, sodium molybdate dihydrate0.2 g/L, boric acid 0.02 g/L, cobalt chloride 0.5 g/L, zinc chloride 20g/L, ferrous sulfate heptahydrate 65 g/L, biotin 0.2 g/L, and sulfuricacid 5 mL/L. The bioreactor process control parameters were set asfollows: Agitation 950 rpm, airflow 1.35 standard liter per minute,temperature 28° C. and pH was controlled (at 6) using ammoniumhydroxide. No oxygen supplementation was provided.

Fermentation cultures were grown for approximately 12 to 16 hours at 28°C. until the initial glycerol was consumed, which was detected by arapid increase in the concentration of dissolved oxygen, referred to asthe DO spike. Feed addition was then initiated at a rate of 15 gglucose/l/hr for 8 hrs. Approximately 8 hrs after feed addition wasinitiated, the feed addition rate was decreased to 13 g glucose/l/hr forthe duration of the fermentation. Also, RQ control was initiated at thistime using agitation feedback control with a minimum agitation of 500rpm and a maximum agitation of 950 rpm to maintain a RQ set point of1.12. The feed was comprised of the following components: yeast extract50 g/L, dextrose anhydrous 500 g/L, magnesium sulfate heptahydrate 3g/L, and PTM1 trace metals 12 mL/L. Optionally, sodium citrate dihydrate(1.66 g/L) was also added to the feed.

After feed initiation, the culture temperature was rapidly shifted to30° C. for one culture of each of the two expressing strains.Additionally, for two cultures of each of the two strains, controlcultures were maintained at 28° C., i.e., without temperature shift. Thetotal fermentation time was approximately 86 hours.

Samples were collected from each culture throughout the fermentation andwhole broth titers were determined and were plotted in arbitrary unitswhich are consistent among the figures in this application.Additionally, at the end of the run, antibody purity was determined(after Protein A purification) by size-exclusion chromatography(SE-HPLC) using the methods described in Example 11.

Results

Ab-B was expressed from engineered P. pastoris strains containing 3copies of the light chain-encoding gene and 3 copies of the heavychain-encoding gene (H3/L3) or 3 copies of the light chain-encoding geneand 4 copies of the heavy chain-encoding gene (H4/L3). Cultures wereinitially grown at 28° C. with glycerol as a carbon source. Afterexhaustion of the glycerol, a continuous glucose feed was initiated andthe culture temperature was rapidly shifted upward to 30° C. which wasmaintained for the duration of the culture, or maintained at 28° C. as acontrol.

At baseline, the H4/L3 strain (FIG. 48A) expressed a higher antibodytiter than the H3/L3 strain (FIG. 48B), with the average final wholebroth titer for unshifted cultures (i.e., maintained at 28° C.) being12% higher for the H4/L3 strain.

The H4/L3 culture shifted to 30° C. exhibited a further 28% increase infinal titer (relative to the average titer from the two H4/L3 culturesmaintained at 28° C., i.e., without a shift). For the H3/L3 strains, theobserved increase in final yield was somewhat less pronounced, however,the yield from the H3/L3 strain shifted to 30° C. increased by about 5%relative to the average final yield from two H3/L3 cultures maintainedat 28° C. (i.e., without a shift).

FIG. 52 shows the temperature of each culture plotted versus time inculture. As expected, upon the temperature shift the cells rapidlyreached the new set point temperature of 30° C., and both the shiftedand unshifted cultures maintained their set point temperaturesthroughout the culture.

Antibody purity was also assessed from each culture using SE-HPLC. Asdescribed in Example 11, the non-reduced samples permitted detection ofabundance of aberrant complexes (the 75 kDa “half-antibody” containingone heavy and one light chain and the HHL complex containing two heavychains but only one light chain). The fraction of protein contained inthe full antibody (“Main peak IgG”) was increased for the two samplesshifted to 30° C. compared to the average of their respective unshiftedcontrols, which resulted from a decrease in both the 75 kDA HL speciesand Prepeak HHL species in the shifted samples relative to the averageof the unshifted samples. Specifically, for the unshifted H4/L3 samplethe average fraction of protein contained in the full antibody peak was74.39%, in the Prepeak HHL was 4.26%, and the 75 kD HL was 12.65%,compared to 83.44%, 4.75%, and 6.15% for the shifted H4/L3 sample,respectively. Likewise for the unshifted H3/L3 samples the averagefraction of protein contained in the full antibody peak was 82.80%, inthe Prepeak HHL was 5.31%, and the 75 kD HL was 4.76%, compared to91.63%, 2.94%, and 1.44%, respectively. In conclusion, the non-reducingSEC analysis demonstrated that the temperature shift (1) increased theaverage amount of antibody contained in the full antibody peak, and (2)decreased the average amount of the aberrant complexes in three out offour instances.

Also as described in Example 11, the reduced samples permitted detectionof the relative abundance of protein contained in the full-length heavyand light chains as well as product-associated variants which wereobserved in three discrete elution peaks. Unlike the observed decreasein aberrant complexes observed with the non-reduced analysis, thereduced samples did not show a consistent improvement in the amount ofantibody contained in the full-length heavy and light chains. Overall,for the two strains the average relative abundance of the heavy chainwas increased by about 1-3% in the shifted cultures compared to theaverage of the unshifted cultures, and the average relative abundance ofthe light chain was unchanged or decreased by about 0.9% for the twostrains.

Example 13

This example tests the effect of a temperature shift on yield and purityof antibodies expressed from P. pastoris. Antibody yield was increasedby 47% on average by an upward temperature shift effected duringculture.

Methods

Ab-A was expressed from a P. pastoris strain containing 4 integratedcopies of the heavy chain gene and 3 integrated copies of the lightchain gene as described in Example 11, except that five cultures weremaintained at 28° C. (i.e., unshifted) and four cultures were shifted to30° C. As in Example 11, the temperate shift was effected, if at all, ata time five minutes after feed initiation. The total fermentation timewas approximately 87 hours.

Using the method described in Example 11, samples were collected fromeach culture throughout the fermentation and whole broth titers weredetermined and were plotted in arbitrary units which are consistentamong the figures in this application, and antibody purity wasdetermined for each culture at the final time point (after Protein Apurification) by size-exclusion chromatography (SE-HPLC) performed onreduced and non-reduced samples.

Results

P. pastoris engineered to express Ab-A was grown in cultures maintainedat 28° C. during an initial growth phase with glycerol as a carbonsource. After exhaustion of the glycerol, a continuous glucose feed wasinitiated and the culture temperature was rapidly shifted upward to anew set-point of 28° C. which was maintained for the duration of theculture (N=4). Five control cultures were maintained at 28° C. (i.e.,nonshifted).

Whole broth antibody titer was determined by periodic sampling and isshown graphically in FIG. 50 (arbitrary units). As depicted, each of thefour cultures that were shifted to 30° C. produced a higher final titerthan all of the nonshifted cultures maintained at 28° C. On average, thefinal titer was 47% higher for the shifted cultures than non-shiftedcultures.

Additionally, purity was assessed by SE-HLPC and compared for theshifted and non-shifted cultures. The non-reduced samples revealed anapproximately 2% increase in the relative amount of protein contained inthe main antibody peak on average, with the prepeak HHL being reduced onaverage by about 21% and the 75 kDa HL peak being increased by 57%. Thereduced samples revealed an across-the-board improvement in purity anddecrease in average relative abundance of all three impurity peaks.Specifically relative to the non-shifted samples, the shifted samplesexhibited a 26% decrease in the RT 9.80 peak, a 74% decrease in the RT10.16 peak, and a 70% decrease in the RT 10.80 peak. In conclusion, thenon-reducing SEC analysis demonstrated that the temperature shift (1)increased the average amount of antibody contained in the full antibodypeak, and (2) decreased the average amount of two out of three aberrantcomplexes. The reduced SEC analysis revealed large decreases in relativeabundance of each of the three detected impurity peaks.

Based thereon, it is concluded that an upward temperature shift from 28°C. to 30° C. (i.e., by 2° C.) reproducibly increased final antibodyyield by an average of 25-30%. Furthermore the purity of the antibodiesas reflected by Capillary Electrophoresis Analysis also reflected anincrease in purity in the temperature shifted cultures when compared tounshifted cultures, more noted in the reduced comparisons.

The above description of various illustrated embodiments of theinvention is not intended to be exhaustive or to limit the invention tothe precise form disclosed. While specific embodiments of, and examplesfor, the invention are described herein for illustrative purposes,various equivalent modifications are possible within the scope of theinvention, as those skilled in the relevant art will recognize. Theteachings provided herein of the invention can be applied to otherpurposes, other than the examples described above.

The invention may be practiced in ways other than those particularlydescribed in the foregoing description and examples. Numerousmodifications and variations of the invention are possible in light ofthe above teachings and, therefore, are within the scope of the appendedclaims.

These and other changes can be made to the invention in light of theabove detailed description. In general, in the following claims, theterms used should not be construed to limit the invention to thespecific embodiments disclosed in the specification and the claims.Accordingly, the invention is not limited by the disclosure, but insteadthe scope of the invention is to be determined entirely by the followingclaims.

Certain teachings related to methods for obtaining a clonal populationof antigen-specific B cells were disclosed in U.S. Provisional patentapplication No. 60/801,412, filed May 19, 2006, and U.S. PatentApplication Pub. No. 2012/0141982, the disclosure of each of which isherein incorporated by reference in its entirety.

Certain teachings related to humanization of rabbit-derived monoclonalantibodies and preferred sequence modifications to maintain antigenbinding affinity were disclosed in International Application No.PCT/US2008/064421, corresponding to International Publication No.WO/2008/144757, entitled “Novel Rabbit Antibody Humanization Methods andHumanized Rabbit Antibodies”, filed May 21, 2008, the disclosure ofwhich is herein incorporated by reference in its entirety.

Certain teachings related to producing antibodies or fragments thereofusing mating competent yeast and corresponding methods were disclosed inU.S. patent application Ser. No. 11/429,053, filed May 8, 2006, (U.S.Patent Application Publication No. US2006/0270045), the disclosure ofwhich is herein incorporated by reference in its entirety.

The entire disclosure of each document cited herein (including patents,patent applications, journal articles, abstracts, manuals, books, orother disclosures), including each document cited in the Background,Summary, Detailed Description, and Examples, is hereby incorporated byreference herein in its entirety.

What is claimed is:
 1. A Pichia pastoris yeast culture which comprises(i) a culture medium and (ii) Pichia pastoris yeast cells which havebeen engineered to comprise nucleic acids which encode for a full lengthhuman or humanized antibody, which nucleic acids are operably linked toat least one promoter and signal sequence that provides for theexpression and secretion of the full length human or humanized antibodyencoded by said nucleic acids, and said at least one promoter thatprovides for the expression of said full length antibody is/are nottemperature inducible, wherein: (1) said Pichia pastoris yeast cellcontaining culture has been cultured by a culture method that hasresulted in the expression and secretion of the full length antibodyinto the culture medium comprising said Pichia pastoris yeast cells, (2)said culture method comprises (a) culturing said Pichia pastoris yeastcells culture at a first temperature which is between 27.5° C. and 28.5°C.; and (b) subsequently culturing said Pichia pastoris yeast cells at asecond temperature is between 30° C. and 31° C., thereby producing aPichia pastoris yeast culture wherein the culture medium comprises thesecreted full length antibody at an increased antibody yield and furthercomprises a decreased amount of antibody-associated variants relative toan otherwise identical culture comprising the same Pichia pastoris yeastcells which have been cultured in the same medium and under the sameconditions, except that culturing is effected entirely at a temperaturewhich is between 30° C. and 31° C. or entirely at a temperature ofbetween 27.5° C. and 28.5° C.; (3) said culture steps (a) and (b) areeffected after an inoculum expansion step; and (4) wherein said yeastculture medium comprising said full-length antibody and said Pichiapastoris yeast cells has not been treated by any purification methodafter said culture method is effected.
 2. The Pichia pastoris yeastculture of claim 1, wherein said Pichia pastoris yeast culture comprisesa decreased relative abundance of any or all of antibody proteins havingaberrant disulfide bonds, antibody proteins having reduced cysteines,and antibody proteins having aberrant glycosylation relative to anotherwise identical Pichia pastoris culture comprising the same Pichiapastoris yeast cells which have been cultured in the same culture mediumand under the same culture conditions, except that said culturing iseffected entirely at a temperature which is between 30° C. and 31° C. orentirely at a temperature of between 27.5° C. and 28.5° C.
 3. The Pichiapastoris yeast culture of claim 1, wherein step (a) comprises culturingsaid Pichia pastoris cells in a culture medium comprising glycerol as acarbon source until said glycerol is exhausted.
 4. The Pichia pastorisyeast culture of claim 1, wherein step (b) comprises a fed batch phase.5. The Pichia pastoris yeast culture of claim 1, wherein step (b)further comprises maintaining the respiratory quotient (RQ) of thePichia pastoris culture at a specified value or in a specified range. 6.The Pichia pastoris yeast culture of claim 5, wherein said respiratoryquotient is between 1.0 and 1.24.
 7. The Pichia pastoris yeast cultureof claim 1, wherein the Pichia pastoris yeast cells comprise 3 or 4integrated copies of the heavy chain gene of the encoded antibody and 3integrated copies of the light chain gene of the encoded antibody,further wherein said integrated copies of the heavy and light chaingenes are integrated into one or more genomic loci selected from thegroup consisting of the pGAP locus, 3′ AOX TT locus; PpURA5; OCH1; AOX1;HIS4; GAP; pGAP; 3′ AOX TT; ARG; the HIS4 TT locus, and one or morerandom chromosomal loci.